CA2998859A1 - Compositions and methods for inducing and enhancing an immune response against infections, diseases, and disorders - Google Patents

Abstract

The present invention relates to compositions and methods for modulating immune responses using at least one cycli di-nucleotide synthetase enzyme gene. Such compositions may be combined with a number of other therapeutics which target modulating immune responses, as well as, treatments that include immune events.

Description

COMPOSITIONS AND METHODS FOR INDUCING AND ENHANCING AN
IMMUNE RESPONSE AGAINST INFECTIONS, DISEASES, AND DISORDERS
Cross-Reference to Related Anulications This application claims the benefit of priority to U.S. Provisional Application No.
62/219,387, filed 16 September 2015, the entire contents of said application is incorporated herein in its entirety by this reference.
Statement of Rights This invention was made with government support under AI057153 and AI105499 awarded by the National Institutes of Health. The government has certain rights in the invention.
Background of the Invention With a limited number of adjuvants approved for human administration, there is a pressing need for the development and testing of vaccine adjuvants that can improve the efficacy and maintain the safety profile of vaccines against resilient infectious diseases and cancers (Alving, CR et al. (2012) Curr Opin Immunol 24: 310-315). The addition of adjuvants to vaccine formulations can serve to significantly improve vaccine efficacy when using less immunogenic antigens (Vessely, C etal. (2009) Journal of pharmaceutical sciences 98: 2970-2993), to decrease vaccine toxicity by diminishing the need for higher vaccine dosages, or reduce the need for repeated boosting (Ahmed, SS etal.
(2011) Science translational medicine 3: 93rv92).
Many significant cellular functions in bacteria, including regulation of motile/sessile phenotypes, virulence capabilities, and global gene expression are mediated by the second messenger bis-(3'-5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) (Gomelsky, M
(2012) J Bacteriol 194: 911-913). C-di-GMP is generated by diguanylate cyclase (DGC) enzymes combining two guanosine-5'-triphosphate (GTP) molecules (Krasteva, PV
et al.
(2012) Protein Sci 21: 929-948). In the mammalian cytosol, the presence of c-di-GMP
molecules can be detected by nucleotide sensors, including absent in melanoma

2 (AIM2) (Jones, JW etal. (2010)Proc Natl Acad Sci USA 107: 9771-9776), the DEAD box-containing helicase (DDX41) (Parvatiyar, K et al. (2012)Nat Immunol 13: 1155-1161), and stimulator of interferon genes (STING), each of which directly binds to c-di-GIVIP, resulting in the increased expression of type I interferons (IFNs) and other innate immune responses (Burdette, DL and R. E. Vance (2013) Nat Immunol 14: 19-26; McWhirter, SM et al.
(2009)J Exp Med 206: 1899-1911).
The direct administration of c-di-GMP has been shown to induce innate immune responses that can enhance protection of mice against challenges with Klebsiella pneumoniae (Karaolis, DK et al. (2007) Infect Immun 75: 4942-4950), Staphylococcus aureus (Brouillette, E et al. (2005) Antimicrob Compositions Chemother 49:
3109-3113), methicillin-resistant S. aureus (MRSA) (Hu, DL et al. (2009) Vaccine 27: 4867-4873), Bordetella pertussis (Elahi, S et al. (2014) PLoS One 9: el09778), Streptococcus pneumoniae (Yan, H et al. (2009) Biochem Biophys Res Commun 387: 581-584), and avian influenza A/H5N1 (Pedersen, GK et al. (2011) PLoS One 6: e26973; Svindland, SC
et al.
(2013) Influenza Other Respir Viruses 7:1181-1193). Specifically, following intranasal challenge with B. pertu.ssis in BALB/c mice, c-di-GMP induced production of cytokines such as TF'N-7, TNF-a, IL-6, and the chemokine MCP-1 in lung tissue (Elahi, S
et al. (2014) PLoS One 9: e109778). Recently, the ability of c-di-GMP to cause robust induction of IFN-f3 has been shown to attenuate experimental autoimmune encephalitis (EAE) progression and onset through the induction of T regulatory (Treg) cells, which suppress helper/effector T cell responses (Huang, L et al. (2013)J Immunol 191: 3509-3513; Lemos, H
eta!, (2014) J Immuno1192: 5571-5578).
The ability of c-di-GMP to trigger mammalian inflammatory responses has recently been harnessed for potential use as a promising vaccine adjuvant (Karaolis, D
K. et at (2007) J Immunol 178: 2171-2181). Several studies suggest that inclusion of c-di-GMP in vaccine formulations can improve vaccine efficacy so as to provide immune protection against various bacterial infections (Elahi, S et al. (2014) PLoS One 9:
e109778; Fatima, M
et al. (2013) Pauli Sci 92: 2644-2650), and cancers (Miyabe, H etal. (2014)J
Control Release 184: 20-27; Chandra, D et al. (2014) Cancer Immunol Res 2: 901-910;
Ohkuri, T et al. (2014) Cancer Immunol Res 2: 1199-1208). Local co-administration (intranasal and sublingual) of H5N1 virosomes and c-di-GMP to BALB/c mice resulted in strong specific B cell and T cell adaptive immunity, but the intramuscular (i.m.) route of vaccination resulted in significantly less protection (Pedersen, GK et al.
(2011) PLoS One 6: e26973). A liposome-based delivery system that improved c-di-GMP cell uptake in vivo resulted in IFN-f3 induction and enhanced tumor-specific cytotoxic T cell activity associated with regression of tumor growth in mice (Miyabe, H etal. (2014) J Control Release 184:

20-27). However, this study suggests that pure extracellular c-di-GMP does not efficiently enter target cells.
Because c-di-GMP activates a robust immune response, there has been ongoing focus on using c-di-GMP as an adjuvant to improve vaccine efficacy (Chen WX
etal.
(2010) Vaccine 28:3080-3085) and delivering or synthesizing c-di-GMP directly within host cells to stimulate innate immunity. Adjuvants are compounds administered alongside vaccine antigens for the purpose of enhancing the longevity, potency, or reducing the effective dose of the antigen without introducing toxic side effects. This is accomplished by stimulating the innate arm of the immune system, resulting in increased cytokine and chemokine production and upregulation of proinflammatory genes (Mosca F et al.
(2008) Proc. Natl. Acad. Sci. U S. A. 105:10501-10506), which then enhances antigen recognition and response (Coffman RL et at. (2010) Immunity 33:492-503). The development of novel adjuvants may be critical to the success of vaccines targeting diseases for which vaccinations have previously failed such as Clostridium difficile, human immunodeficiency virus, malaria, and cancer. Despite the demand, currently there are few adjuvants approved for human use. The most commonly used adjuvants are aluminum-salt (Alum)-based;
however, these adjuvants suffer drawbacks including local reactions to administration, inadequate T-cell responses, allergic IgE-type responses, and are ineffective with specific types of antigens (Gupta RK (1998) Adv. Drug Deily. Rev. 32:155-172). Other less-commonly utilized adjuvants include oil and water emulsions, lipopolysacharide derivatives, self-assembling viral nanoparticles, and cholera toxin B subunit (Gupta RK
(1998) Adv. Dnig Deily. Rev. 32:155-172). While each adjuvant offers different advantages and disadvantages, there is a large demand for novel adjuvants and compositions that can be paired with and improve vaccine antigens.
In addition to cyclic di-GMP, additional cyclic di-nucleotides have similarly been shown to bind to eukaryotic cytoplasmic receptors, such as STING, to stimulated a Type-I
interferon response. Cyclic di-AMP (c-di-AMP) is an additional second messenger synthesized in bacteria by diadenylate cyclase (DAC) domain containing enzymes that has important roles in cell-wall and metabolic homeostatis (Commichau F.M. et at.
(2015) Mol Microbiol. (2):189-204). C-di-AMP is secreted by invasive bacterial pathogens such as Listerisa monocytogenes and Chlatnydia trachomatis to upregulate inflammatory responses via STING (Barker JR et al. (2013) MBio. 4(3):e00018-13; Woodward JJ et. al.
(2010) Science 328(5986):1703-5). A third cyclic di-nucleotide, cyclic GMP-AMP
(cGAMP),

- 3 -which is synthesized by both bacteria and eukaryotes in a different isomeric form, also activates STING-dependent inflammation. cGAMP was first shown to be synthesized by the enzyme DncV in the bacterial pathogen Vibrio cholerae (Davies B.W. et. al.
(2012) Cell. 149(2):358-70) where it controls chemotaxis and intestinal colonization.
cGAMP has not been widely studied, but a recent report indicates that it is important in exoelectrogenesis in Geobacter species (Nelson J.W. et. al. (2015) Proc Nati Acad Sci 112(17):5389-94). All bacterial cyclic di-nucleotides including c-di-GMP, c-di-AMP, and cGAMP exists as cyclic rings with two 3'-5' phosphodiester linkages. Recently, the eukaryotic protein cGAS, which is well known to activate Type I interferon pathways in response to cytoplasmic DNA, was shown to synthesize cGAMP with a mixed ring linkage of 2'-5' and 3'-5' (cGAMP-ML) (Sun L. et. al. (2013) Science. 339(6121):786-91; Gao P.
(2013) Cell. 153(5):1094-107). cGAMP-ML then binds to STING to induce inflammation.
All of these cyclic di-nucleotides are capable of inducing Type I interferon responses in a STING-dependent manner (Yi G. et al. (2013) PLoS One. 8(10): e77846).
Summary of the Invention The present invention is based, at least in part, on a novel platform to produce cyclic di-nucleotides (e.g., c-di-GMP, c-di-AMP, cGAMP) inside host cells, as an adjuvant to exploit a host-pathogen interaction, that is useful in upregulating, initiating, enhancing, or stimulating an immune response to thereby treat conditions that would benefit from upregulating an immune response (e.g., pathogenic infections and cancers). In one aspect, provided herein are compositions of matter comprising a vector (e.g., any gene therapy vector, including but not limited to, all adenovirus serotypes, similar vectors derived from AAV, retroviruses, lentiviruses, and DNA based vectors, AdVCA0956, or AdVCA0848) having at least one cyclic di-nucleotide synthetase enzyme gene (e.g., DAC, DncV, Hypr-GGDEF, cGAS, DisA, DGCs, Vibrio cholerae DGCs, such as VCA0956 or VCA0848).
Numerous embodiments are described herein that can be applied to any aspect of the present invention or embodiment thereof For example, in one embodiment, pharmaceutical compositions, vaccines, and adjuvants comprising the vectors of the present invention, are provided. In another embodiment, co-administration of a combination vaccine comprising the vectors of the present invention and an extracellular antigen (Ag) (e.g., viral-associated antigen, bacterial-associated antigen, tumor-associated antigen, such as ovalbumin, Clostridium difficile-derived Toxin B or Toxin A, or HIV-1 derived Gag

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antigen), is provided. Any of the aforementioned compostions, when introducted in vitro and in vivo, markedly increases cycli di-nucleotide (e.g., c-di-GMP, cGAMP) levels and stimulates immune responses (e.g., the innate, adaptive, or humoral immune response).
One aspect of the invention relates to a vector comprising at least one cyclic di-nucleotide synthetase enzyme gene. In some embodiments, the vector is a gene-therapy vector. In another embodiment, the vector is selected from the group consisting of adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus. In yet another embodiment, the vector is a DNA-based vector. In some embodiments, the vector is an adenoviral vector. In another embodiment, the vector is a replication defective adenoviral vector. In yet another embodiment, the at least one cyclic di-nucleotide synthetase enzyme gene is derived from a bacterial, fungal, protozoal, viral, or pathogenic strain. In some embodiments, the at least one cyclic di-nucleotide synthetase enzyme gene is derived from a bacterial strain. In another embodiment, the bacterial strain is Vibrio cholerae. In some embodiments, the at least one cyclic di-nucleotide synthetase enzyme gene is selected from the group consisting of diadenylate cyclase (DAC), DncV, Hypr-GGDEF, DisA, cGAS, and diguanylate cyclase (DGC). In another embodiment, the at least one cyclic di-nucleotide synthetase enzyme gene is DGC. In yet another embodiment, the DGC comprises a sequence which is at least 80% identical to the sequences set forth in Table 1. In some embodiments, the DGC gene is VCA0956 gene. In another embodiment, the VCA0956 gene comprises a nucleotide sequence which is at least 80% identical to SEQ ID
NO: 33.
In yet another embodiment, the DGC gene is VCA0848 gene. In some embodiments, the VCA0848 gene comprises a nucleotide sequence which is at least 80% identical to SEQ ID
NO: 68. In another embodiment, the vector comprises an adenovirus selected from non-human, human adenovirus serotype, or any adenovirus serotype developed as a gene transfer vector. In still another embodiment, the non-human adenovirus comprises an adenovirus selected from chimp, equine, bovine, mouse, chicken, pig, or dog.
In some embodiments, the adenovirus is human adenovirus serotype 5. In some embodiments, the adenovirus has at least one mutation or deletion in at least one adenoviral gene. In another embodiment, the adenoviral gene is selected from the group consisting of El A, E IB, E2A, E2B, E3, E4, L1, L2, L3, L4, and 1,5. In yet another embodiment, the adenovirus has a deletion in ElA, E IB, and E3, or combinations thereof. In some embodiments, the at least

- 5 -one cyclic di-nucleotide synthetase enzyme gene is operatively linked to a transcriptional and translational regulatory sequences.
Another aspect of the invention relates to a combination comprising any of the aforementioned vectors. In some embodiments, the combination comprises at least one therapeutic agent. In some embodiments, the agent is another vaccine, an immunemodulatory drug, a checkpoint inhibitor, or a small molecule inhibitor.
In some embodiments, the combination further comprises a therapy for immune events. In some embodiments, the therapy is irradiation.
Yet another aspect of the invention relates to a pharmaceutical composition comprising any of the aforementioned vectors, and a pharmaceutically acceptable composition selected from the group consisting of excipients, diluents, and carriers. In some embodiments, the pharmaceutical composition comprises the vector at a purity of at least 75%.
Still another aspect of the invention relates to an adjuvant comprising any of the aforementioned vectors. Another aspect of the invention relates to a vaccine comprising any of the aforementioned vectors, any of the aforementioned pharmaceutical compositions, or any of the aforementioned adjuvants. In some embodiments, the vaccine further comprises an antigen. In some embodiments, the antigen is provide in a second adenoviral vector. In yet some embodiment, the antigen is immunogenic. In some embodiments, the antigen is an extracellular antigen. In still another embodiment, the antigen is a viral-associated antigen, pathogenic-associated antigen, protozoal-associated antigen, bacterial-associated antigen, fungal antigen, or tumor-associated antigen. In some embodiments, the antigen is selected from the group consisting of Ovalbumin (OVA)-specific, HIV-1-derived Gag Ag, Clostridium difficile-derived toxin B, and Clostridium Offici/e-derived toxin A.
Yet another aspect of the invention relates to a method of inducing or enhancing an immune response in a mammal, comprising: administering to the mammal a pharmaceutically effective amount of any of the aforementioned vaccines such that the immune response is enhanced or stimulated.
Another aspect of the invention relates to a method of treating a mammal having a condition that would benefit from upregulati on of an immune response comprising administering to the subject a therapeutically effective amount of any of the aforementioned vaccines such that the condition that would benefit from upregulation of an immune response is treated. In some embodiments, the method further comprises administering one

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7 or more additional compositions or therapies that upregulates an immune response or treats the condition. In another embodiment, the one or more additional compositions or therapies is selected from the group consisting of anti-viral therapy, immunotherapy, chemotherapy; radiation, and surgery. In yet another embodiment, the condition that would benefit from upregulation of an immune response is selected from the group consisting of cancer, a viral infection, a bacterial infection, fungal infection, and a protozoan infection.
In still another embodiment, the immune response is the innate immune response, adaptive immune response, or humoral immune response. In some embodiments, the vaccine increases or stimulates cyclic di-GMP (c-di-GMP), cyclic di-AMP (c-di-AMP), cyclic GMP-AMP (cGAMP), any cyclic di-nucleotide, or combinations therof,levels in said mammal. In another embodiment, the vaccine increases or stimulates the secretion of cytokines and chemokines. In yet another embodiment, the cytokines and chemokines are selected from the group consisting of IFN-fl, IL-la, IL-4, IL-6, IL12-p40, IFN-y, G-CSF, Eotaxin, KC, MCP-1, MIP-la, MIP-10, and RANTES. In still another embodiment, the vaccine increases or stimulates an immune response selected from the group consisting of DC maturation, NK cell response, T-cell response, and B-cell reponse, or combination thereof. In some embodiments, the immune response increases the population of immunce cells selected from the group consisting of CD86+ CD1le+CD11b-DCs, CD69+
NK1.1+
CD3 NK cells, CD69 CD19+ CD3'B cells, CD69+ CD3+ CD8' T cells, and CD69+ CD3+

CD8+ T cells, or combinations thereof. In another embodiment, the subject is a mammal.
In some embodiments, the mammal is an animal model of the condition. In yet another embodiment, the mammal is a human. In some embodiments, the mammal is an avian species. In still another embodiment, the avian species is G. gallus, or eggs derived therefrom. In some embodiments, the vaccine is administered intradermally, intramuscularly, intraperitoneally, intratumorally, peritumoroally, retroorbiatlly, or intravenously via injection. In another embodiment, the vaccine is administered concomitantly or conjointly. In yet another embodiment, the first vector comprising the cyclic di-nucleotide synthetase enzyme gene lowers the effective dose for the second vector comprising the antigen. In still another embodiment, the administration is repeated at least once. In some embodiments, the effective amount is from about lx106vp to about 5x1011 vp. In another embodiment, the effective amount is from about 1x106vp to about 5x109vp.
In yet another embodiment, the effective amount is about 1x106 vp, about lx VP) about 1x108vp, or about 5x109 vp, In some embodiments, the effective amount is about 5x109 vp. In another embodiment, the effective amount is about lx101 , about 0.5x10", about lx1011, about 2x1011, about 3x10", about 4x1011, or about 5x1011 viral particles (vp). In some embodiments, the effective amount is about 2x1011 vp. In yet another embodiment, the effective amount is about 10 pg/mL, about 20 pg,/mL, about 30 p.g/mL, about 40 pg/mL, about 50 pg/mL, about 60 ug/mL, about 70 ps/mL, about 80 g/mL, about pg/mL, about 100 g/mL, about 125 pg/mL, about 150 pg/mL, about 175 pg/mL, and ug/mL. In some embodiments, the effective amount is about 100 pg/mL.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Brief Description of Figures Figure 1 contains 2 panels, identified as panels A and B, depicting LC-MS/MS
used to quantify c-di-GMP in HeLa cells. Panel A shows that HeLa cells were transfected with plasmid vectors containing the VCA0956 allele or the active site mutant allele, VCA0956*.
Bars represent the mean of 5 independent cultures. Panel B shows c-di-GMP in HeLa cells cultured in T75 flasks and transfected with plasmid vectors containing the VCA0956 allele at 24 and 48 hours. Bars represent the mean of independent cell cultures (24 hours, N=3; 48 hours, N=2).
Figure 2 depicts HeLa cells infected with 500 M.O.I. Ad5 vectors. Bars represent the mean of 3 independent cultures; error bars indicate standard deviation. bd indicates below detection.
Figure 3 contains 2 panels, identified as panels A and B, depicting infection of Ad5-VCA0956 in a murine system. Panel A shows that after 24 hours qPCR was used to quantify Ad5 genomes in liver cells (black) or spleen cells (checkered). Data were normalized to internal GADPH control. Panel B depicts LC-MS/MS was used to quantify c-di-GMP extracted from the liver (black) or spleen (checkered). Bars represent the mean of 3 independent mouse samples; error bars indicate standard deviation. bd indicates below detection. Panel B depicts that in the presence of rIFNg, 72.9% of the cells was PE
positive.

- 8 -Figure 4 contains 3 panels, identified as panels A, B and C, depicting qRT-PCR
of mouse liver gene transcripts 24 hours after infection with Ad5 vectors. The data were normalized to internal GADPH control. Fold change indicates each value normalized to values measured from mock treated mice. Results are separated into liver gene expression increased by Ad5-VCA0956 (Panel A), decreased by Ad5-VCA0956 (Panel B), or unaffected by Ad5-VCA0956 (Panel C). Bars represent the mean of 3 independent mouse samples; error bars indicate standard deviation. Brackets indicate statistical significance, which was determined using a two-tailed Student's t-test (P <0.05).
Figure 5 depicts IFN-fl concentrations in the plasma of mice infected with Ad5 vectors. Mice were infected with either Ad5-Null (stripes), Ad5-VCA0956 (black), or Ad5-VCA0956* (grey). At 6 and 24 hours, IFN-f3 was quantified from plasma samples.
Brackets indicate statistical significance, which was determined using a one-way ANOVA
test combined with a Bonferroni posttest (** p < 0.01). Bars indicate the mean of independent mouse plasma samples (n=2: Mock, Ad5-Null; n=3: Ad5-VCA0956, Ad5-VCA0956*) and error bars indicate standard deviation. bd indicates below detection.
Figure 6 contains 12 panels, identified as panels A, B, C, D, E, F, G, H, I, J, K, and L, depicting plasma cytokine and chemokine levels in mice infected with Ad5 vectors.
Mice were infected with either Ad5-Null (stripes), Ad5-VCA0956 (black), or Ad5-VCA0956* (grey). At 6 and 24 hours, cytokines and chemokines were quantified from plasma samples. Brackets indicate statistical significance, which was determined using a two-way ANOVA test combined with a Bonferroni posttest (* p <0.05; ** p <0.01). Bars indicate the mean of independent mouse plasma samples Mock, Ads-Null; n=3: Ad5-VCA0956, Ad5-VCA0956*) and error bars indicate standard deviation. IL-la (Panel (A)), IFN-y (Panel (B)), MCP-1 (Panel (C)), IL-4 (Panel (D)), G-CSF (Panel (E)), MW-la (Panel (F)), IL-6 (Panel (G)), Eotaxin (Panel (H)), MIP-43 (Panel (I)), IL-12p40 (Panel (J)), KC
(Panel (K)), and RANTES (Panel (L)).
Figure 7 contains two panels, identified as panels A and B, depicting C.
difficile TA-specific IgG from the plasma of mice I.M. vaccinated with (A) 1 x 107 vp Ad5-TA and Ad5-VCA0956 or (B) 5 x 109 vp Ad5-TA and Ad5-VCA0956 (both 14 d.p.i.) was quantified using an ELISA assay. The 013450 was measured at various plasma dilutions.
Each point represents the mean of 6 independent mouse plasma samples, and error bars indicate standard deviation.

- 9 -Figure 8 shows IFN-y ELISPOT analysis of mice vaccinated with Ad5-TA and Ad5 vectors. Mice were administered (I.M.) varying doses of both Ad-TA and either Ad-VCA0956 (black) or Ad-VCA0956* (grey). After 14 days, splenocytes were ex vivo stimulated with a C. df fficile specific peptide and the number of IFNy secreting splenocytes was determined using ELISPOT. Each point represents an individual mouse. Lines indicate the mean of the replicates, and error bars indicate standard error. *
indicates statistical significance using a two-way ANOVA test combined with a Bonferroni posttest (P
< 0.05).
Figure 9 shows that active VCA0848 produces significant amounts of c-di-GMP in mice. Male 6-8 weeks old BALB/c WT mice were retro-orbitally i.v. injected with 2x109 vps/mouse of AdVCA0848 (n=3); or 2x 1011 vps/mouse of AdVCA0848't (n=3) or AdVCA0848 (n=3). As a control not injected (neves) mice (n=2) were included.
At 24 hpi mice were sacrificed and liver samples were collected, and immediately snap frozen in liquid nitrogen. 20 mg of liver samples were used for c-di-GMP extraction as described in methods section. C-di-GMP production measurements were performed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Bars represent mean SD from different groups. Statistical analysis was completed using One Way ANOVA followed by a Student-Newman-Keuls post-hoc test. A value of p<0.05 was deemed statistically significant. "bd' , below detection.
Figure 10 contains 6 panels, identified as panels A, B, C, D, E, and F, depicting that AdVCA0848 stimulates strong induction of IFN-13 and activates innate and adaptive immune cells. Male 6-10 weeks old C57BL/6 WT mice (n=4) were i.v. injected (retro-orbitally) with lx101 vps/mouse of AdNull, AdVCA848, or not injected (naive) as control.
At 6 hpi mice were sacrificed and spleens and blood samples were obtained.
Panel A shows an ELISA-based assay to determine the amount of IFN-13 produced in plasma (diluted 1:2) from naive, mice injected with AdNull, AdVCA0848. Splenocytes harvested and FACS
analysis conducted as described in methods and materials. Effects of AdNull and AdVCA0848 (with representative results) on the activation of CD86+CD11c+CD11b-DCs (Panel B), CD69+ NK1.1+ CD3- NK cells (Panel C), CD69+ CD194CD3" B cells (Panel D), CD69+CD3+ CDS T cells (Panel E), and CD69+ CD3+ CD8+ T cells (Panel F). Bars with the indicated colors represent mean SD. Statistical analysis was completed using One Way ANOVA followed by a Student-Newman-Keuls post-hoc test. A value of p<0.05 was deemed statistically significant. The (**) and (***) denote significance over naive animals p<0.05 and p<0.001, respectively.

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Figure 11 contains 4 panels, identified as panels A, B, C, and D, depicting that AdVCA0848 enhances OVA-specific adaptive T cell responses. Male 6-10 weeks old C57BL/6 mice (n=5) were injected with OVA alone, OVA + AdVCA0848, OVA +
AdNull, or not injected as described in materials and methods. At 14 dpi, mice were sacrificed and splenocytes atl x106 cells/well were ex vivo stimulated with MHC class 1-restricted OVA-derived peptide SIINFEKL, OVA protein, heat-inactivated Ad5 particles, or with only media (unstimulated). The ELISPOT assays for IFN-7 (Panels A and B) and IL-2 (Panels C and D) were performed. Bars with the indicated colors represent mean SD
for samples stimulated with the indicated stimulations. Results are representative of two independent experiments. Statistical analysis was completed using One Way ANOVA
followed by a Student-Newman-Keuls post-hoc test. A value of p<0.05 was deemed statistically significant. The (**) and (***) denote significance over naïve animals p<0.05 and p<0.001, respectively.
Figure 12 contains 4 panels, identified as panels A, B, C, and D, depicting that AdVCA0848 enhances OVA-specific adaptive B cell responses. Male 8-10 weeks old C57BL/6 mice (n=5) were injected with OVA + AdNull, OVA + AdVCA0848õ or not injected (naïve) as described in materials and methods. Panels A and B show that at 6 dpi, mice were retro-orbitally bleeded to determine OVA and Ad5-specific B cell response by ELISA-based measurement for total IgG with the indicated plasma dilutions.
Panels C and D shows that at 14 dpi, mice were sacrificed; blood samples obtained, and plasma samples were prepared and used for ELISA-based measurement for total OVA and Ad5-specific IgG with the indicated plasma dilutions. Bars with the indicated colors represent mean SD for samples from different groups. Results are representative of two independent experiments. Statistical analysis was completed using One Way ANOVA followed by a Student-Newman-Keuls post-hoc test. A value of p<0.05 was deemed statistically significant.
Figure 13 contains 2 panels, identified as panels A and B, depicting that co-injecting AdVCA0848 and AdGag results in significant inhibitory effects of Gag-specific T
cell responses. Female 6-8 weeks old BALB/c mice (n=4) were i.m. co-injected in the tibialis anterior with viral particles of AdGag (5x106vps/mouse) along with 3 different doses (5x 107 , 5x 108, or 5x109 vps/mouse) of either AdNull or AdVCA0848, in the presence of an uninjected group of mice as control naive. At 14 dpi, mice were sacrificed and splenocytes (at 5x105 cells/well) were ex vivo stimulated with the 15-mer HIV/Gag-

- 11 -derived immunogenic peptides AMQ (Panel A), or with UV-inactivated adenoviruses (Panel B) for the IFNI ELISPOT assays as described in materials and methods.
Bars with the indicated colors represent mean SD. Results are representative of two independent experiments. Statistical analysis was completed using One Way ANOVA followed by a Student-Newman-Keuls post-hoc test. A value of p<0.05 was deemed statistically significant. The (**) and (***) denote significance over naïve animals p<0.05 and p<0.001, respectively. The (a) denote significance over AdVCA0848 at the dose of 5x109vps/mouse (p<0.05).
Figure 14 contains 3 panels, identified as panels A, B, and C, depicting that co-injecting AdVCA0848 and AdGag results in significant inhibitory effects of Gag-specific CD8+T cells. Female 6-8 weeks old BALB/c mice (n=4) were i.m. co-injected in the tibialis anterior with viral particles of AdGag (5x106vps/mouse) along with 3 different doses (5x 107 , 5x108, or 5x109vps/mouse) of either AdNull or AdVCA0848, in the presence of an uninjected group of mice as control naive. At 14 dpi, mice were sacrificed and splenocytes harvested and used at 1x106 cells/well for tetramer staining using PE-labeled MHC class I tetramer folded with the AMQ peptide as described in materials and methods followed by FACS analysis for Tee" Gag-specific CD8+ T cells (Panel A). Multi-parameter staining was conducted to determine the overall frequency of IFN-y (Panel B) and TNF-a (Panel C) producing CD8+ T cells followed by FACS analysis conducted on BD
LSRII flow cytometer as described in methods and materials. Results are representative of two independent experiments. Bars with the indicated colors represent mean SD.
Statistical analysis was completed using One Way ANOVA followed by a Student-Newman-Keuls post-hoc test. A value of p<0.05 was deemed statistically significant. The (**) and (***) denote significance over naïve animals p<0.05 and p<0.001, respectively.
The (a) denote significance over AdVCA0848 dose of 5x109 vps/mouse (p<0.05).
Figure 15 contains 4 panels, identified as panels A, B, C, and D, depicting that co-injecting AdVCA0848 resulted in significant inhibition of Gag and ToxB-specific B cell response. Female 6-8 weeks old BALB/c mice (n=4) were i.m. co-injected in the tibialis anterior with the indicated viral injections and as described in materials and methods of AdVCA0848 along with either AdGag or AdToxB in the presence of uninjected mice control naives. At 14 dpi, mice were sacrificed and plasma samples collected.
Total IgG
levels of Gag-specific (plasma dilution 1:25) antibodies (Panel A) or Ad5-specific (plasma dilution 1:400) (Panel B) were measured to determine the effect of indicated does of

- 12 -AdVCA0848 on Gag-specific B cell response by ELISA. ELISA was also used to determine the effect of AdVCA0848 on ToxB-specific (Panel C) and Ad5-specific (Panel D) B cell response by measuring total IgG levels at the indicated plasma dilutions. Results are representative of two independent experiments. Bars with the indicated colors represent mean SD. Statistical analysis was completed using One Way ANOVA followed by a Student-Newman-Keuls post-hoc test. A value of p<0.05 was deemed statistically significant. The (**) and (***) denote significance over naive animals p<0.05 and p<0.001, respectively.
Figure 16 shows co-administration of AdGag and AdVCA0848 does not inhibit the translation of Gag protein. Male 6-8 weeks old BALB/c WT mice were retro-orbitally iv.
injected with 1x10111 ¨11 tu vps/mouse of AdGag alone (n=3), or co-injected with lx10"
vps/mouse AdVCA0848 (n=4), AdNull (n=3), or not injected (naives) (n=3) as control.
Figure 17 shows that AdVCA0848 produces significant amounts of c-di-GMP in mice which surpasses that produced by AdVCA0956. Male 6-8 weeks old BALB/c WT
mice were retro-orbitally iv. injected with 2x1011 vps/mouse of AdVCA0956 (n=4), AdVCA0848 (n=4), AdNull (n=3), or not injected (naives) (n=3) as control. At 24 hpi mice were sacrificed and liver samples were collected, and immediately snap frozen in liquid nitrogen. 20 mg of liver samples were used for c-di-GMP extraction as described in methods section. C-di-GMP production measurements were performed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Bars represent mean SD from different groups. Statistical analysis was completed using One Way ANOVA followed by a Student-Newman-Keuls post-hoc test. A value of p<0.05 was deemed statistically significant. "bd' , below detection.
Figure 18 contains 6 panels, identified as panels A, B, C, D, E, and F, depicting that active VCA0848 stimulates strong induction of IFN-13 and activates innate and adaptive immune cells. Male 6-10 weeks old C57BL/6 WT mice (n=3) were retro-orbitally iv.
injected with lx1010 vps/mouse of AdVCA0848'`, AdVCA848, or not injected (naive) as control. At 6 hpi mice were sacrificed and spleens and blood samples were obtained. Panel A shows an ELISA-based assay to determine the amount of IFN-I3 produced in plasma (diluted 1:2) from naive, mice injected with AdVCA0848"'", or AdVCA0848.
Splenocytes harvested and FACS analysis conducted as described in methods and materials.
Effects of AdVCA08481nut or AdVCA0848 (with representative results) on the activation of CD11c+CD11b-DCs (Panel B), CD69+ NK1.1+ CD3-NK cells (Panel C), CD69+ CD19+

- 13 -CD3- B cells (Panel D), CD69+ CD3+ CD8 T cells (Panel E), and CD69+ CD3+ CD8+
T
cells (Panel F). Bars with the indicated colors represent mean SD.
Statistical analysis was completed using One Way ANOVA followed by a Student-Newman-Keuls post-hoc test. A
value of p<0.05 was deemed statistically significant Figure 19 shows that AdVCA0848 enhances OVA-specific adaptive B cell responses when co-injected with OVA. Male 8-10 weeks old C57BL/6 mice (n---5) were injected with OVA alone, OVA + AdNull, OVA + AdVCA0848, or not injected (naive) as described in materials and methods. At 14 dpi, mice were sacrificed; blood samples obtained, and plasma samples were prepared and used for ELISA-based measurement for total OVA and Ad5-specific IgG (plasma dilution 1:1000). Bars with the indicated colors represent mean SD for samples from different groups. Results are representative of two independent experiments. Statistical analysis was completed using One Way ANOVA
followed by a Student-Newman-Keuls post-hoc test. A value of p<0.05 was deemed statistically significant. The (**) and (***) denote significance over naïve animals p<0.05 and p<0.001, respectively.
Figure 20 contains 3 panels, identified as panels A, B, and C, depicting that active VCA0848 results in significant inhibitory effects of Gag-specific T cell and B
cell responses and significant enhancement of Ad5-specifc T cell and B cell response by AdVCA0848 and AdGag co-administration. Female 6-8 weeks old BALM mice (n=3) were i.m, co-injected in the tibialis anterior with viral particles of AdGag (5x106 vps/mouse) along with 5x109 vps/mouse of either AdVCA0848't or AdVCA0848, in the presence of an uninjected group of mice as control naive (n=2). At 14 dpi, mice were sacrificed and peripheral blood and spleens were collected. Panel A shows that splenocytes (at lx 106 cells/well) were ex vivo stimulated with the 15-m er HIV/Gag-derived immunogenic peptides AMQ or with UV-inactivated adenoviruses for the IFN-y ELISPOT
assays as described in materials and methods. Total Gag-specific (Panel B), or Ad5-specific (Panel C) IgG levels at the indicated plasma dilutions were measured to determine the effect of indicated does of AdVCA0848 and AdVCA0848't on Gag-specific B
cell response by ELISA. Bars with the indicated colors represent mean SD.
Statistical analysis was completed using One Way ANOVA followed by a Student-Newman-Keuls post-hoc test. A value of p<0.05 was deemed statistically significant.
Figure 21 depicts the conserved protein domain for COG2199 (GGDEF domain, diguanylate cyclase (c-di-GMP synthetase) or its enzymatically inactive variants) provided

- 14 -from http ://www. ncbi.nlm. nih.gov/Structure/cdd/cddsrv. cgi?as cbi n=88zmax al n=10& seltype=2&
uid----COG2199.
Figure 22 depicts a sequence alignment of various DncV homologs from bacteria (from Figure S1 of Kranzusch PJ et al. (2014) Cell 158(5):1011-21).
Figure 23 lists the putative HYPR domains in Geobacter and Pelobacter and identifies the conserved residues. The bottom sequence (ccPleD/1-454) is a known GGDEF
from Caulobacter crescentus for comparison.
Note that for every figure containing a histogram, the bars from left to right for each discreet measurement correspond to the figure boxes from top to bottom in the figure legend as indicated.
Detailed Description of the present invention The present invention is based, at least in part, on a novel approach to produce cyclic di-nucleotides (e.g., c-di-GMP, c-di-AMP, c-di-GAMP, or others) inside host cells as an adjuvant to exploit a host-pathogen interaction and initiate an innate immune response.
Provided herein are compositions of matter comprising a vector (e.g., any gene therapy vector) having at least one cyclic di-nucleotide synthetase enzyme. As described herein, in some embodiments, c-di-GMP can be synthesized in vivo by transducing a diguanylate cyclase (DGC) gene (e.g., Vibrio cholerae DCGs, such as VCA0956 or VCA0848), into mammalian cells using a non-replicating adenovirus serotype 5 (Ad5) vector (e.g., AdVCA095 or AdVCA0848). Expression of the DGC led to the production of c-di-GMP
in vitro and in vivo, and this was able to alter pro-inflammatory gene expression in murine tissues and increase the secretion of numerous cytokines and chemokines when administered into animals. Co-expression of the DGC modestly increased T-cell responses to a Clostridium difficile antigen expressed from an adenovirus vaccine. This adenovirus c-di-GMP delivery system offers a novel method to administer c-di-GMP as an adjuvant to stimulate innate immunity during vaccination. In some embodiments, AdVCA0848 is more potent than AdVCA0956 and produces elevated amounts of c-di-GMP when expressed in mammalian cells in vivo. As described herein, this novel platform improves induction of type I interferon p (FFN-13) and activation of innate and adaptive immune cells early after administration into mice as compared to control vectors. Co-administration of the extracellular antigen (e.g., protein ovalbumin (OVA)) and AdVCA0848 adjuvant

- 15 -significantly improved OVA-specific T cell responses as detected by EFN-y and ELISPOT, while also improving OVA-specific humoral B cell adaptive responses.
The data presented herein confirm that in vivo synthesis of cyclic di-nucleotides (e.g., c-di-GMP) stimulates strong innate immune responses that correlate with enhanced adaptive immune responses to concomitantly administered extracellular antigen, which can be utilized as an adjuvant to heighten effective immune responses for protein-based vaccine platforms against pathogenic infections and cancers. An extension of the compositions and methods decribed herein is to similarly express other cyclic di-nucleotide synthetase enzymes such as those containing a DAC domain (Hengge R. et. al. (2016)J
Bacteriol.
198(1):15-26), DncV or other enzymes that synthesis bacterial cGAMP (Davies B.W. et. al.
(2012) Cell. 149(2):358-70), Hypr-GGDEF enzymes that can make c-di-GMP, c-di-AMP, or cGAMP (Hallberg Z.F. et. al. (2016) Proc Natl Acad Sci 113(7):1790-5.), or cGAS (Sun L. et. al. (2013) Science 339(6121):786-91; Gao P (2013) Cell. 153(5):1094-107).
I. Definitions The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the gra more than one element. mmatical object of the article. By way of example, "an element" means one element or more than one element.
As used herein, "adenoviruses" are DNA viruses with a 36-kb genome. There are human adenovirus serotypes that have been distinguished on the basis of their resistance to neutralization by antisera to other known adenovirus serotypes. Adenoviruses as used herein encompass non-human or any adenovirus serotype developed as a gene transfer vector. Non-human adenovirus comprises an adenovirus selected from chimp, equine, bovine, mouse, chicken, pig, dog, or any mammalian or non-mammalian species.
Although the majority of adenoviral vectors are derived from serotypes 2 and 5, other serotypes may also be used. The wild type adenovirus genome is divided into early (El to E4) and late (L1 to L5) genes, e.g., El A, ElB, E2A, E2B, E3, E4, Li, L2, L3, L4, or L5.
Adenovirus vectors can be prepared to be either replication competent or non-replicating.
Replication defective adenoviral vectors may comprise at lease one deletion of any of the El to E4 or Li to L5 genes. Replication deficient adenovirus based vectors are described in Hartman ZC et al. (2008) Virus Res. 132:1-14. In some embodiments, the replication defective adenovirus comprises deletions of the El and E3 genes. Foreign genes can be inserted into three areas of the adenovirus genome (El, E3, or E4) as well as behind the major late

- 16 -promoter. The ability of the adenovirus genome to direct production of adenoviruses is dependent on sequences in El.
Adenovirus vectors transduce large fragments of DNA into a wide range of cells in order to synthesize proteins in vivo, and gene expression can be modulated and even localized to specific cell types. Unlike other types of viral delivery systems, DNA delivered by adenovirus vectors does not integrate into the genome and thus circumvents the danger of insertional mutagenesis (Aldhamen YA et al. (2011)Front. Immun. 2:1-12).
Adenovirus vectors have been shown to induce innate immunity, which is partially due to inducing the STING DNA recognition pathway (Lam Eel al. (2013)J. Virol. 88:974-981).
Additionally, adenovirus vectors can be produced cost-efficiently in high abundance.
Importantly, adenovirus vectors are currently being used in human clinical trials world-wide (Fukazawa Tel al. (2010) Int. J. Mol. Med 25:3-10).
The term "adjuvant" is used in its broadest sense as any substance or composition (e.g., AdVCA0848 or AdVCA0956) which enhances, increases, upwardly modulates or otherwise facilitates an immune response to an antigen be it added exogenously or already present such as a tumor associated antigen. The immune response may be measured by any convenient means such as antibody titre or level of cell-mediated response.
The term "body fluid" refers to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g., amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, peritoneal fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit). In a one embodiment, body fluids are restricted to blood-related fluids, including whole blood, serum, plasma, and the like.
The terms "cancer" or "tumor" or "hyperproliferative disorder" refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer is generally associated with uncontrolled cell growth, invasion of such cells to adjacent tissues, and the spread of such cells to other organs of the body by vascular and lymphatic menas. Cancer invasion occurs when cancer cells intrude on and cross the normal boundaries of adjacent tissue, which can be measured by assaying cancer cell migration, enzymatic destruction of basement

- 17 -membranes by cancer cells, and the like. In some embodiments, a particular stage of cancer is relevant and such stages can include the time period before and/or after angiogenesis, cellular invasion, and/or metastasis. Cancer cells are often in the form of a solid tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, WaldenstrOm's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma;
leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelornonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia);
and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple

- 18 -myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer whose phenotype is determined by the method of the present invention is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, brenner, or undifferentiated. In some embodiments, the present invention is used in the treatment, diagnosis, and/or prognosis of melanoma and its subtypes.
The term "coding region" refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues, whereas the term "noncoding region"
refers to regions of a nucleotide sequence that are not translated into amino acids (e.g., 5' and 3' untranslated regions).
The term "complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

- 19 -More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
The term "control" refers to any reference standard suitable to provide a comparison. In one embodiment, the control comprises obtaining a "control sample" from which expression product levels are detected and compared to the expression product levels from the test sample. Such a control sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient or healthy patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a healthy patient or the cancer patient, cultured primary cells/tissues isolated from a subject such as a normal subject or the cancer patient, adjacent normal cells/tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a healthy subject, or a primary cells/tissues obtained from a depository. In another embodiment, the control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment (for example, standard of care cancer therapy). It will be understood by those of skill in the art that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the present invention.
The term "cycli-di-nucleotides," or c-di-nucleotides as used herein encompasses any cyclic di-nucleotides, including but not limted to, c-di-GMP, c-di-AMP, or cGAMP. C-di-nucleotides have been shown to bind to eukaryotic cytoplasmic receptors, such as STING, to stimulated a Type-I interferon response. All bacterial cyclic di-nucleotides including c-di-GMP, c-di-AMP, and cGAMP exists as cyclic rings with two 3'-5' phosphodiester linkages.
The term "cyclic di-AMP" refers to a specific bacterial second messenger synthesized in bacteria that has important roles in cell-wall and metabolic homeostatis (Commichau F.M. et. al. (2015) Mol Microbiol. (2):189-204). C-di-AMP has also been shown to be an essential singalnig molecule in Staphylococcus aureus (Corrigan R.M.
(2013) Proc Nall Acad Sci 110(22):9084-9) and Listeria monocytogenes (Commichau F.M.
(2015) Mol Microbiol 97(2):189-204). C-di-AMP is secreted by invasive bacterial

- 20 -pathogens such as Listerisa monocytogenes and Chlamydia trachomatis to upregul ate inflammatory responses via STING (Barker JR et. al. (2013) MBio. 4(3):e00018-13;
Woodward JJ et. al. (2010) Science 328(5986):1703-5).
The term "cyclic di-GMP," or "c-di-GMP" as used herein is is a bacterial specific second messenger that controls a wide range of phenotypes including motility, biofilm formation, and virulence (Romling U etal. (2013)Microbiol. Mot Biol. Rev. 77:1-52). C-di-GMP was first discovered in 1987 by Benziman et al. (Ross P etal. (1987) Nature 325:279-281), and since has been predicted to be utilized in >75% of all bacteria in representatives from every major bacterial phyla (Seshasayee ASN etal. (2010) Nucleic Acids Res. 38:5970-5981). Diguanylate cyclase enzymes (DGCs) which contain conserved GGDEF domains synthesize c-di-GMP from two GTP molecules. In contrast, c-di-GMP is hydrolyzed by c-di-GMP specific phosphodiesterase enzymes (PDEs) which contain conserved EAL or HD-GYP domains (Romling U etal. (2013) Microbial Mol. Biol.
Rev.
77:1-52). Bacteria typically contain numerous DGCs and PDEs within their genomes; for example, the marine bacterium Vibrio cholerae encodes 70 predicted c-di-GMP
turnover domains (Galperin MY et al. (2001) FEMS Microbial. Lett 203:11-21).
Previous studies indicate that c-di-GMP is a potent stimulator of innate immunity in eukaryotic organisms. This occurs at least in part through the protein STING, which senses pathogen derived nucleic acids in the cytoplasm and subsequently activates a signaling cascade to stimulate a type-I interferon response (McWhirter SM et al. (2009)1 Exp. Med.
206:1899-1911; Sauer JD et al. (2011) Infect. Immun. 79:688-694). Studies show that the presence of c-di-GMP can trigger the production of IL-2, IL-4, 1L-5, IL-6, IL-8, IL-12p40, IL-17, IP-10, TNF-a, KC, MIP- 1 a, M1P-113, MIP-2, MCP-1, RANTES, IFN-f3, stimulate the NLRP3 inflammasome pathway, and promote the recruitment and activation of macrophages, NK cells, af3 conventional T cells, and enhance DC maturation (Sauer JD
et al. (2011) Infect. Immun. 79:688-694; Ebensen Tel al. (2007) Vaccine 25:1464-1469;
Abdul-Sater AA etal. (2013) EMBO reports 14:900-906; Ebensen T et al. (2007) Clin.
Vaccine Immunol. 14:952-958; Karaolis DKR et al. (2007)1 Immunol. 178:2171-2181;
Karaolis DKR et al. (2007) Infect. Immun. 75:4942-4950; Yan HE et al. (2009) Biochem.
Biophys. Res. Commun. 387:581-584; Gray PM etal. (2012) Cell Immunot 278:113-119;
Blaauboer SM etal. (2014)1 Immzinol. 192:492-502). Furthermore, in vivo studies have shown that co-administration of purified c-di-GMP with an antigen confers increased protection of animals in several different murine challenge models, including those utilizing

- 21 -Staphylococcus aureus, Klebsiella pneumoniae, and Streptococcus pneumoniae (Karaolis DKR etal. (2007) J. Immunol. 178:2171-2181; Karaolis DKR etal. (2007) Infect.
Immun.
75:4942-4950; Yan FIB et al. (2009) Biochem. Biophys. Res. Commun. 387:581-584;
Ogunniyi AD etal. (2008) Vaccine 26:4676-4685).
The term "cyclic GMP-AMP" (cGAMP) refers to a second messenger produced by both bacteria and eukaryotic cells (designated as cGMAP-ML). cGAMP has not been extensively studied in bacteria, but it has been shown to regulate virulence and chemotaxis in the bacterial pathogen Vibrio choelrae (Davies B.W. et. al. (2012) Cell.
149(2):358-70) and evidence suggests it could regulate exoelectrogenesis in Geobacter species (Nelson J.W. et. al. (2015) Proc Natl Acad Sci 112(17)5389-94) although this has not been fully demonstrated. All bacterial cyclic di-nucleotides including c-di-GMP, c-di-AMP, and cGAMP exists as cyclic rings with two 3'-5' phosphodiester linkages. Recently, the eukaryotic protein cGAS, which is well known to activate Type I interferon pathways in response to cytoplasmic DNA, was shown to synthesize cGAMP with a mixed ring linkage of 2'-5' and 3'-5' (cGAMP-ML) (Sun L. et. al. (2013) Science. 339(6121):786-91; Gao P.
(2013) Cell. 153(5):1094-107). cGAMP-ML then binds to STING to induce inflammation.
The term "cyclic di-nucleotide synthetase enzyme" as used herein refers to a class of enzymes which synthesizes cyclic-di nucleotides, including but not limited to, c-di-AMP, c-di-GMP, or cGAMP. Such cyclic di-nucleotide synthetase enzymes include but are not limited to diguanylate cyclase (DGC), Hypr-GGDEF, diadenylate cyclase (DAC), DncV, cGAS, and DisA (c-di-AMP synthesis). As noted in Burroughs AM et al. (2015) Nucleic Acids Res. 43(22):10633-54: "All synthetases that use NTPs as substrates to generate the above-mentioned cyclic and linear nucleotides belong to just four distinct superfamilies.
The classical adenylyl and guanyly1 cyclases (Mock M. etal. (1991)].
Bacteriol.
173:6265-6269) and GGDEF domains which generate c-di-GMP (Pei J. et. al.
(2001) Proteins 42:210-216) belong to a large superfamily of enzymes that also includes most DNA polymerases, reverse transcriptases, viral RNA-dependent RNA polymerases and T7-like DNA-dependent RNA polymerases Another distinct, large superfamily of nucleotidyltransferases, also including DNA polymerase 13 (polB superfamily) (Aravind L.
et al. (1999) Nucleic Acids Res. 27:1609-1618; Kuchta K. et al. (2009) Nucleic Acids Res.
37:7701-7714), contains several nucleotide-generating families; namely the CyaA-like bacterial adenylyl cyclases (Mock M. et a/.(1991) J. Bacteriol 173:6265-6269;
Aravind L.
et al. (1999) Nucleic Acids Res. 27:1609-1618), the cyclic 2'-5' GMP-AMP
synthase

- 22 -, W02017/049127 PCT/US2016/052198 (cGAS), bacterial 3'-5 cGAMP synthetases typified by the V.cholerae DncV
(formerly known as VC0179) (Davies. B.W. et al. (2012) Cell 149:358-370; Kato K. et al.
(2015) Structure 23:843-850) and 2'-5'A synthetase (oligoadenylate synthetase: OAS).
The characterized c-di-AMP synthetases belong to the DisA superfamily, members of which directly monitor DNA integrity via a fused DNA-binding domain (Bejerano-Sagie M. et al.
(2006) Cell 125:679-69; Witte G. et al. (2008) Mol. Cell 30:167-178;
Oppenheimer-Shaanan Y. et. al (2011) ELL60 Rep. 12:594-601; Campos S.S. et al. (2014)].
Bacteriol. 196:568-578)."
Cyclic di-nucleotide synthetase enzyme genes may encompass those derived from any of the V cholerae strains, including but not limited to, 01 str. C6706 Contig_56 (Accession: NZ_AHGQ01000056.1 GI: 480994251); 01 str. C6706 Contig_20 (Accession:
NZ_AHGQ01000020.1 GI: 480994215); 01 str. C6706 Contig_30 (Accession:
NZ_AHGrQ01000030.1 GI: 480994225); 01 str. C6706 Contig_42 (Accession:
NZ_AHGQ01000042.1 GI: 480994237); 01 str. C6706 Contig_40 (Accession:
NZ AHGQ01000040.1 GI: 480994235); 01 str. C6706 Contig_37 (Accession:
NZ AHGQ01000037.1 GI: 480994232); 01 str. C6706 Contig_36 (Accession:
NZ_AHGQ01000036.1 GI: 480994231); 01 str. C6706 Contig_62 (Accession:
NZ_AHGQ01000062.1 GI: 480994257); 01 str. C6706 Contig_27 (Accession:
NZ AHGQ01000027.1 GI: 480994222); 01 biovar El Tor str. N16961 chromosome I
(Accession: NC 002505.1 GI: 15640032); 01 biovar El Tor str. N16961 chromosome (Accession: NC 002506.1 GI: 15600771); 2012EL-2176 chromosome 2 (NZ_CP007635.1 GI: 749293683); 2012EL-2176 chromosome 1 (Accession: CP007634.1 GI:
695931389);
TSY216 chromosome 1 (Accession: CP007653.1 GI: 861210305); strain ATCC 25874 CFSAN20.contig.1 (Accession: LRIK01000002.1 GI: 977936890); strain ATCC 11629 CFSAN19.c,ontig.4 (Accession: LOSM01000005.1 GI: 967485342); YB1A01 YBOl_AOl_contig_l (Accession: LBCL01000001.1 GI: 940519882); YB2G05 YB02_G05_contig_7 (Accession: LBFZ01000007.1 GI: 940550115); InDRE 4262 chromosome I Chrl_contig7 (Accession: JZUB01000007.1 GI: 769091410); InDRE

chromosome I Chrl_contig7 (Accession: JZUA01000007.1 GI: 769088978); YB8E08 YB08_E08_contig_18 (Accession: LBGN01000018.1 GI: 940599519); YB7A06 YB07_A06_contig_3 (Accession: LBGL01000003.1 GI: 940598755); YB7A09 YBO7 _ A09 _ contig _12 (Accession: LBGM01000012.1 GI: 940597590); YB6A06 YB06_A06_contig_1 1 (Accession: LBGKO1000011.1 GI: 940592937); YB5A06

- 23 -YB05_A06_contig_7 (Accession: LBGJO1000007.1 GI: 940588968); YB4G05 YB04_G05_contig_14 (Accession: LB0001000014.1 GI: 940577186); YB4F05 YB04_F05_contig_14 (Accession: LBGF01000014.1 GI: 940572881); YB4B03 YB04_B03_contig_3 (Accession: LBGD01000003.1 GI: 940570625); YB4C07 YB04_C07_contig_32_consensus (Accession: LBGE01000031.1 GI: 940565209);
YB3B05 YB03_1305_contig_2 (Accession: LBGB01000002.1 GI: 940562726); YB2G07 YB02_G07_contig_1 (Accession: LBGA01000001.1 GI: 940559910); YB1G06 Y80I_G06_contig_1 (Accession: LBFV01000001.1 GI: 940544222); YB2A05 YB02_A05_contig_14 (Accession: LBFW01000014.1 GI: 940540732); M1522 contig00012 (Accession: LQCA01000012.1 GI: 974047169); M988 contig00008 (Accession: LQBX01000008.1 GI: 974034339); 01 biovar El Tor strain FJ147 (Accession:
CP009042.1 GI: 785752771); 2740-80 chromosome 2 (CP016325.1); 01 str. KW3 chromosome II (CP006948.1); TSY216 chromosome 2 (CP007654.1); 01 biovar El Tor strain FJ147 chromosome II (CP009041.1); 2012EL-2176 chromosome 2 (CP007635.1);
MS6, chromosome 2 (AP014525.1); 01 str. 2010EL-1786 chromosome 2 (CP003070.1);

MJ-1236 chromosome 2 (CP001486.1); 0395 chromosome II (CP001236.1); M66-2 chromosome II (CP001234.1); 0395 chromosome 1(CP000626.1); 01 biovar eltor str.
N16961 chromosome II (AE003853.1); IEC224 chromosome II (CP003331.1); LMA3894-4 chromosome 1I (CP002556.1); 1154-74 (CP010811.1); or 10432-62 (CP010812.1).
Cyclic di-nucleotide synthetase enzyme genes may also encompass those derived from any species, for example, but not limited to, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter junk Acinetobacter Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter radioresistens, Actinobacillus lignieresii, Actinobacillus suis, Aeromonas ca viae, Aeromonas hydrophila, Aeromonas veronii subsp. sobria, Aggregatibacter actinomycetemcomitans, Arcobacter butzleri, Arcobacter nitrofigilis, Bacillus amyloliquefaciens, Bacillus anthracis, Bacillus bataviensis, Bacillus cellulosilyticus, Bacillus cereus, Bacillus clausii, Bacillus licheniformis, Bacillus megateriztm, Bacillus pumilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroidesfragilis, Bordetella avium, Bordetella bronchiseptica, Bordetella pertusis, Bordetella petrii, Brucella abortus, Bruce/la melitensis, Bruce/la suis, Burkholderia cenocepacia, Burkholderia mallei, Burkholderia multivorans, Burkholderia pseudomallei, Burkholderia thailandensis, Campylobacter concisus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp. venerealis, Campylobacter gracilis,

- 24 -Campylobacter hominis, Campylobacter jejuni, Campylobacter rectus, Campylobacter showae, Campylobacter upsaliensis, Citrobacter freundii, Citrobacter koseri, Clostridium asparagiforme, Clostridium botulinum, Clostridium butyricum, Clostridium difficile, Clostridium perfringens, Clostridium saccharobutylicum, Clostridium tetani, Corynebacterium diphtheriae, Cotynebacterium pseudotuberculosis, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Escherichia coil, Fusobacterium necrophorum, Fusobacterium nucleatum, Granulicatella adiacens, Granulicatella elegans, Haemophilus equigenitalis, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus paragallinarum, Haemophilus parasuis, Haemophilus pleuropneumoniae, Haemophilus somnus, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae, Legionella oakridgensis, Legionella pneumophila, Leptospira biflexa, Leptospira illni, Leptospira interrogans, Listeria monocytogenes, Lysinibacillus fusiformis, Lysinibacillus sphaericus, Moraxella bovis, Morganella morganii, Mycobacterium abscesses, Mycobacterium africanum, Mycobacterium avium, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Plesiomonas shigelloides, Propionibacterium acnes, Proteus hanseri, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella cholerasuis, Salmonella enterica subsp.
enterica, Salmonella enteritidis, Salmonella paratyphi, Salmonella oiphi, Serratia plymuthica, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus epidermidis, Staphylococcus equonim, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus pasteuri, Staphylococcus pettenkgferi, Staphylococcus pseudointermedius, Staphylococcus saprophyticus, Staphylococcus simiae, Staphylococcus simulans, Staphylococcus warner!, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus dysgalactiae subsp. equisimilis, Streptococcus equi, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus uberis, Streptococcus zooepidermicus, Taylorefta asinigenitalis, Taylorella equigenitalis, Treponema carateum, Treponema cuniculi, Treponema hyodisenteriae, Treponema pallidum, Treponema suis, Veillonella alypica, Veillonella dispar, Veillonella parvula, Veillonella rail!, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificans, Yersinia enterocolitica, Yersinia pestis and Yersinia pseudotuberculosis.

- 25 -, WO 2017/049127 The term "cGAS" refers a cytoplasmic eukaryotic receptor that responds to cytoplasmic DNA to produced cGAMP-ML (Sun L. et. al. (2013) Science.
339(6121):786-91; Gao P. (2013) Cell. 153(5):1094-107).
The term DAC refers to "diadenylate cyclase" enzymes encoded in bacteria that synthesis c-di-AMP. Bacteria encode a number of different DAC domain enzymes that may be targeted to the membrane of the cytoplasm (Commichau F.M. (2015)Mol Microbiol.
97(2):189-204). The first described DAC is DisA from Bacillus subtilis designated by C0G1623 (Oppenheimer-Shaanan Y. et. al. (2011) EMBO Rep. 2011 Jun;12(6):594-601).
The term "diguanylate cyclase," or "DGC", unless otherwise specified, refers to known DGC RNA, DNA, and polypeptides, as well as its isoforms, and biologically active fragments thereof. DGC enzymes typically encode GGDEF domain that are described in the COG database as COG2199. V. cholerae encodes upwards of 40 unique DGCs, many of which have been shown to synthesize c-di-GMP in this bacterium (Beyhan, S
etal.
(2008)J Bacteriol190: 7392-7405; Lim, B etal. (2006) Mol Microbiol 60: 331-348;
Beyhan, S et al. (2007)Mol Microbiol 63: 995-1007; Massie, JP etal. (2012) Proc Nall Acad Sc, USA 109(31):12746-51; Hunter, JL et al. (2014) BMC Microbiol 14: 22).
These DGCs have highly divergent c-di-GMP synthesis activities (Shikuma, NJ etal.
(2012) PLoS
Pathog 8: e1002719; Massie, JP etal. (2012) Proc Natl Acad Sci USA
109(31):12746-51).
Approximately half of these DGCs are thought to be integral inner membrane proteins, while the other half are cytoplasmic. Each contains a unique N-terminal sensory domain that is predicted to be regulated by environmental or host derived cues (Galperin, MY
(2004) Environ Microbiol 6: 552-567). Tens of thousands of DGCs have been identified across bacterial genomes (Hunter, JL et al. (2014) BMC Microbiol 14: 22).
Thus, these genes offer a wide-range of unique enzymes possessing different properties that can be transduced by vectors to potentially modulate immune responses. DGC genes may encompass those derived from any of the V cholerae strains listed above, or any of the bacterial sources set forth above. Table 1, the Figures, and the Examples, below provide representative DGC sequences. For example, Table 1 provides DGC sequences encompassed within the scope of compositions-of-matter and methods of the present invention. However, any protein containing a protein domain belonging to the COG family COG2199 is considered a DGC (i.e., COG2199 which is the DGC (i.e., also called a GGDEF) domain that synthesizes c-di-GMP; see

- 26 -

27 PCT/US2016/052198 http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?ascbin=8&maxaln=10&seltype =2&
uid=C0G2199 at Figure 21 and Galperin MY etal. (2015) Nucleic Acids Res.
43(Database issue) D261-9; Ausmees N et al. (2001)Microbio/. Lett. 204(1):163-167; Paul R
et al.
(2004) Genes Dev. 18(6):715-727; Chan C et al. (2004) Proc. Natl. Acad Sci.
U.S.A.
101(49):17084-17089; Ryjenkov DA et al. (2005)J Bacteriol. 187(5):1792-1798;
Aldridge P et al. (1999)Mol. Microbiol. 1999 Apr; 32(2):379-391; Pei J et al. (2001) Proteins 2001 42(2):210-216; Tal R et al. (1998)f. Bacteriol. 180(17):4416-4425; Marcher-Bauer etal.
(2015) Nucleic Acids Res. 43 (Database issue):D222-6).
The term "DncV" refers to a bacterial enzyme encoded in V. cholerae that has been shown to synthesize cGAMP (Davies B.W. et. al. (2012) Cell. 149(2):358-70). As noted in Kranzusch PJ et al. (2014) Cell 158(5):1011-21, in spite of the minimal sequence identity, the results in the paper showed that DncV is both a structural and functional homolog of mammalian cGAS, which demonstrates for the first time a direct connection between the biosynthetic machinery for generating dinucleotide signals in multiple kingdoms of life.
The core of DncV adopts a template-independent nucleotidyl-transferase fold defined by 13 strands 132-5, similar to the originally characterized CCA-adding enzyme (Figure 1) (Xiong et al. (2004) Nature 430, pp. 640-645). In spite of minimal sequence identity (-10%), the overall structure of DncV is remarkably similar to that of human cGAS
(Kranzusch PJ et al.
(2014) Cell 158(5):1011-21). Figure 22 from Kranzusch depicts a sequence alignment of various DncV homologs from bacteria.
The term "Hypr-GGDEF" refers to a certain class of DGC enzymes that have a GGDEF domain that have been shown to synthesize cGAMP depending on the available nucleotide substrates (Hallberg Z.F. et. al. (2016) Proc Nall Acad Sci 113(7):1790-5.). As noted in Hallberg ZF eta! (2016) Proc Natl Acad Sci USA. 113(7):1790-5, hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3', 3'-cGAMP) (see Fig.
S9 (Figure 23 herein) which lists the putative HYPR domains in Geobacter and Pelobacter and identifies the conserved residues. The bottom sequence (ccPleD/1-454) is a known GGDEF from Caulobacter crescentus for comparison).
DisA (c-di-AMP synthesis). NCBI lists the domain as pfam02457: DisA_N
From the NCBI website: "DisA bacterial checkpoint controller nucleotide-binding: The DisA protein is a bacterial checkpoint protein that dimerizes into an octameric complex.
The protein consists of three distinct domains. This domain is the first and is a globular, nucleotide-binding region; the next 146-289 residues constitute the DisA-linker family, pfam10635, that consists of an elongated bundle of three alpha helices (alpha-6, alpha-10, and alpha-11), one side of which carries an additional three helices (alpha7-9), which thus forms a spine like-linker between domains 1 and 3. The C-terminal residues, of domain 3, are represented by family HHH, pfam00633, the specific DNA-binding domain. The octameric complex thus has structurally linked nucleotide-binding and DNA-binding HhH
domains and the nucleotide-binding domains are bound to a cyclic di-adenosine phosphate such that DisA is a specific di-adenylate cyclase. The di-adenylate cyclase activity is strongly suppressed by binding to branched DNA, but not to duplex or single-stranded DNA, suggesting a role for DisA as a monitor of the presence of stalled replication forks or recombination intermediates via DNA structure-modulated c-di-AM13 synthesis."
pfam02457 is a member of the superfamily c110589 (see Marchler-Bauer A et al.
(2015) Nucleic Acids Res. 43 (Database issue):D222-6).
Examples of diseases or conditions wherein enhancement of a protective immune response is desired includes, but are not limited to viral, pathogenic, protozoal, bacterial, or fungal infections and cancer.
Viral infectious diseases include human papilloma virus (HPV), hepatitis A
Virus (HAY), hepatitis B Virus (HBV), hepatitis C Virus (HCV), retroviruses such as human immunodeficiency virus (HIV-1 and HIV-2), herpes viruses such as Epstein Barr Virus (EBV), cytomegalovirus (CMV), HSV-1 and HSV-2, influenza virus, Hepatitis A
and B, FIV, lentiviruses, pestiviruses, West Nile Virus, measles, smallpox, cowpox, ebola, coronavirus, retrovirus, herpesvirus, potato S virus, simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Moloney virus, ALV, Cytomegalovirus (CMV), Epstein Barr Virus (EBV), or Rous Sarcoma Virus (RSV). In addition, bacterial, fungal and other pathogenic diseases are included, such as Aspergillus, Brugia, Candida, Chiluingunya, Chlamydia, Coccidia, Cryptococcus, Dengue, Dirofilaria, Gonococcus, Histoplasma, Leishmania, Mycobacterium , Mycoplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis, P. vivax in Anopheles mosquito vectors, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Toxoplasma and Vibriocholerae. Exemplary species include Neisseria gonorrhea, Mycobacterium tuberculosis, Candida albi cans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vagina/is, Group B Streptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus .
Bruce/la melitensis, Bruce/la suis, Bruce/la canis, Campylobacter fetus, Campylobacter

- 28 -fetus intestinalis, Leptospira pomona, Listeria monocytogenes, Brucella ovis, Chlamydia psittaci, Trichomonas foetus, Toxoplasma gondii, Escherichia coli, Actinobacillus equuli, Salmonella abortus ovis, Salmonella abortus equi, Pseudomona,s aeruginosa, Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis, Mycoplasma bovigenitalium, Aspergillus fumigatus, Absidia ramosa, Trypanosoma equiperdum, Clostridium tetani, Clostridium botulirtum; or, a fungus, such as, e.g., Paracoccidioides brasiliensis; or other pathogen, e.g., Plasmodium fakiparum. Also included are National Institute of Allergy and Infectious Diseases (NIAID) priority pathogens. These include Category A compositions, such as variola major (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularaemia), filoviruses (Ebola hemorrhagic fever, Marburg hemorrhagic fever), arenaviruses (Lassa (Lassa fever), Junin (Argentine hemorrhagic fever) and related viruses); Category B compositions, such as Coxiella burnetti (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), alphaviruses (Venezuelan encephalomyelitis, eastern & western equine encephalomyelitis), ricin toxin from Ricinus communis (castor beans), epsilon toxin of Clostridium perfringens; Staphylococcus enterotoxin B, Salmonella species, Shigella dysenteriae, Escherichia coli strain 0157:H7, Vibrio cholerae, Cryptosporidium parvum; Category C compositions, such as nipah virus, hantaviruses, yellow fever in Aedes mosquitoes, and multidrug-resistant tuberculosis;
helminths, such as Schistosoma and Taenia; and protozoa, such as Leishmania (e.g., L. mexicana) in sand flies, Plasmodium, Chagas disease in assassin bugs.
Other bacterial pathogens include, but are not limited to, bacterial pathogenic gram-positive cocci, which include but are not limited to: pneumococci;
staphylococci; and streptococci. Pathogenic gram-negative cocci include: meningococci; and gonococci.
Pathogenic enteric gram-negative bacilli include: enterobacteriaceae;
pseudomonas, acinetobacteria and eikenella; melioidosis; salmonella; shigellosis;
hemophilus; chancroid;
brucellosis; tularemia; yersinia (pasteurella); streptobacillus moniliformis and spirilum;
listeria monocytogenes; erysipelothrix rhusiopathiae; diphtheria; cholera;
anthrax; and donovanosis (granuloma inguinale). Pathogenic anaerobic bacteria include;
tetanus;
botulism; other clostridia; tuberculosis; leprosy; and other mycobacteria.
Pathogenic spirochetal diseases include: syphilis; treponematoses: yaws, pinta and endemic syphilis;
and leptospirosis. Other infections caused by higher pathogen bacteria and pathogenic fungi include: actinomycosis; nocardiosis; cryptococcosis, blastomycosis, histoplasmosis and

- 29 -coccidioidomycosis; candidiasis, aspergillosis, and mucormycosis;
sporotrichosis;
paracoccidiodomycosis, petriellidiosis, torulopsosis, mycetoma and chromomycosis; and dermatophytosis. Rickettsial infections include rickettsia] and rickettsioses.
Examples of mycoplasma and chlamydial infections include: mycoplasma pneumoniae;
lymphogranuloma venereum; psittacosis; and perinatal chlamydial infections.
Pathogenic protozoans and helminths and infections eukaryotes thereby include: amebiasis;
malaria;
leishmaniasis; trypanosomiasis; toxoplasmosis; pneumocystis carinii;
giardiasis; trichinosis;
filariasis; schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm) infections. While not a disease or condition, enhancement of a protective immune response is also beneficial in a vaccine or as part of a vaccination regimen as is described herein.
The terms "enhance", "promote" or "stimulate" in terms of an immune response includes an increase, facilitation, proliferation, for example a particular action, function or interaction associated with an immune response.
The term "homologous" as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A
first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50%
homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
The term "host cell" is intended to refer to a cell into which any of the nucleotide sequence of the one or more cyclic di-nucleotide synthetase enzyme, or fragment thereof, such as a recombinant vector (e.g., gene therapy vector) of the present invention, has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may

-30-. W02017/049127 occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
As used herein, the term "immune cell" refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B
cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
As used herein, the term "immune response" includes T cell mediated and/or B
cell mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.
The term "immunotherapeutic composition" can include any molecule, peptide, antibody or other composition which can stimulate a host immune system to generate an immune response to a tumor or cancer in the subject.
As used herein, the term "inhibit" includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction. For example, a pathogenic infection or cancer is "inhibited" if at least one symptom of the pathogenic infection or cancer, such as hyperproliferative growth, is alleviated, terminated, slowed, or prevented.
As used herein, cancer is also "inhibited" if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.
As used herein, the term "interaction," when referring to an interaction between two molecules, refers to the physical contact (e.g., binding) of the molecules with one another.
Generally, such an interaction results in an activity (which produces a biological effect) of one or both of said molecules. The activity may be a direct activity of one or both of the molecules. Alternatively, one or both molecules in the interaction may be prevented from binding their ligand, and thus be held inactive with respect to ligand binding activity (e.g., binding its ligand and triggering or inhibiting an immune response). To inhibit such an interaction results in the disruption of the activity of one or more molecules involved in the interaction. To enhance such an interaction is to prolong or increase the likelihood of said physical contact, and prolong or increase the likelihood of said activity.

- 31 -A "kit" is any manufacture (e.g., a package or container) comprising at least one reagent (e.g, gene therapy vector of the present invention, an extracellular Ag) for use in stimulating or enhancing an immune response when adminitered. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.
The term "modulate" includes up-regulation and down-regulation, e.g., enhancing or inhibiting a response.
The term "sample" is typically whole blood, plasma, serum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluid (e.g., as described above under the definition of "body fluids"), or a tissue sample such as a small intestine, colon sample, or surgical resection tissue. In certain instances, the method of the present invention further comprises obtaining the sample from the individual prior to detecting or determining the presence or level of at least one marker in the sample.
The term "synergistic effect" refers to the combined effect of two or more compositions of matter of the present invention that is greater than the sum of the separate effects of the compositions of matter alone.
The term "mammal" refers to any healthy animal, subject or human, or any animal, mammal or human afflicted with a condition of interest (e.g., pathogenic infection or cancer). The term "subject" is interchangeable with "patient."
The term "purity" as used herein, refers to any of compositons or matter described herein which is substantially free of impurities or artifacts that may interfere in the efficacy of the composition when administered. Impurities or artifacts may include interfering antibody, polypeptide, peptide or fusion protein. In one embodiment, the language "purity of at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%" includes preparations of vectors (e.g., gene therapy vectors), or pharmaceutical compositions, vaccines, adjuvants, combination vaccines (e.g., vector combined with an additional therapeutic agent), or the like, having less than about 30%, 20%, 15%, 10%, 5% (by dry weight) of impurities and/or artifacts.
The terms "treatment" "treat" and "treating" encompasses alleviation, cure or prevention of at least one symptom or other aspect of a infection, disorder, disease, illness or other condition (e.g., pathogenic infections or cancer), or reduction of severity of the condition, and the like. A composition of matter of the invention need not affect a complete cure, or eradicate every symptom or manifestation of a disease, to constitute a viable therapeutic composition. As is recognized in the pertinent field, drugs employed as therapeutic compositions may reduce the severity of a given disease state, but need not

- 32 -abolish every manifestation of the disease to be regarded as useful therapeutic compositions. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total, whether detectable or undetectable) and prevention of relapse or recurrence of disease. Similarly, a prophylactically administered treatment need not be completely effective in preventing the onset of a condition in order to constitute a viable prophylactic composition. Simply reducing the impact of a disease (for example, by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood that the disease will occur or worsen in a subject, is sufficient.
"Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. In one embodiment, an indication that a therapeutically effective amount of a composition has been administered to the patient is a sustained improvement over baseline of an indicator that reflects the severity of the particular disorder.
By a "therapeutically effective amount" of a composition of the invention is meant an amount of the composition which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect is sufficient to "treat" the patient as that term is used herein.
As used herein, a vaccine is a composition that provides protection against a pathogenic infection (e.g., protozoal, viral, or bacterial infection), cancer or other disorder or treatment for a pathogenic infection, cancer or other disorder. Protection against a pathogenic infection, cancer or other disorder will either completely prevent infection or the tumor or other disorder or will reduce the severity or duration of infection, tumor or other disorder if subsequently infected or afflicted with the disorder. Treatment will cause an amelioration in one or more symptoms or a decrease in severity or duration.
For purposes herein, a vaccine results from infusion of injection (either concomitantly, sequentially or simultaneously) of an antigen and a composition of matter produced by the methods herein.
As used herein, amelioration of the symptoms of a particular disorder by administration of a particular composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the compositions of matter described herein.

-33-, As used herein a "vaccination regimen" means a treatment regimen wherein a vaccine comprising an antigen and/or any of the gene therapy-vectors (alone or in combination) described herein, as an adjuvant, is administered to a subject in combination, simultaneously, in either separate or combined formulations, or sequentially at different times separated by minutes, hours or days, but in some way act together to provide the desired enhanced immune response to the vaccine in the subject as compared to the subject's immune response in the absence of a composition in accordance with the invention. In some embodiments of the methods described herein, the "antigen"
is not delivered but is already present in the subject, such as those antigens which are associated with tumors. In some embodiments of the compositions described herein, the gene therapy vectors can have activity that is independent of their adjuvant properties.
For example, and by no way limiting, STING activation has been shown to have a direct toxic effect on cancer cells.
As used herein, the term "vector", used interchangeably with "construct", refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA
loop into which additional DNA segments may be ligated. Another type of vector is a viral vector (e.g., replication defective adenovirus, retroviruses, or lentivirus), wherein additional DNA
segments may be ligated into the viral genome. Viral vectors may also include polynucteotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g.
circular); nucleic acid molecules that comprise DNA, RNA, or both, and other varieties of polynucleotides

- 34 -, WO 2017/049127 known in the art. Also included are DNA-based vectors, which can be delivered "naked" or formulated with liposomes to help the uptake of naked DNA into cells.
There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.
GENETIC CODE
Alanine (Ala, A) GCA, GCC, GCG, GOT
Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT
Asparagine (Asn, N) AAC, AAT
Aspartic acid (Asp, D) GAC, GAT
Cysteine (Cys, C) TGC, TGT
Glutamic acid (Glu, E) GAA, GAG
Glutamine (Gin, Q) CAA, CAG
Glycine (Gly, G) GGA, GGC, GGG, GGT
Histidine (His, H) CAC, CAT
Isoleucine (Ile, I) ATA, ATC, ATT
Leucine (Leu, L) CTA, CTC, CTG, OTT, TTA, TTG
Lysine (Lys, K) AAA, AAG
Methionine (Met, M) ATG
Phenylalanine (Phe, F) TTC, TTT
Proline (Pro, P) CCA, CCC, COG, OCT
Serine (Ser, S) AGO, AGT, TCA, TOO, TOG, TOT
Threonine (Thr, T) ACA, ACC, ACG, ACT
Tryptophan (Trp, W) TGG
Tyrosine (Tyr, Y) TAO, TAT
Valine (Val, V) GTA, GTC, GTG, GTT
Termination signal (end) TAA, TAG, TGA
An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide

-35-sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence.
Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.
In view of the foregoing, the nucleotide sequence of a DNA or RNA coding for a protein or polypeptide of the present invention (or any portion thereof) can be used to derive the protein or polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for a protein or polypeptide amino acid sequence, corresponding nucleotide sequences that can encode the protein or polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence).
Thus, description and/or disclosure herein of a nucleotide sequence which encodes a protein or polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, description and/or disclosure of a protein or polypeptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
Finally, nucleic acid and amino acid sequence information for any cyclic di-nucleotide synthetase enzymes (e.g., any DGC, DAC, DncV, cGAS, Hypr-GGDEF, DisA) are well known in the art and readily available on publicly available databases, such as the National Center for Biotechnology Information (NCBI). For example, any protein containing a protein domain belonging to the COG family COG2199 is considered a DGC
(i.e., C0G2199 which is the DGC (i.e., also called a GGDEF) domain that synthesizes c-di-GMP; see http.//www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?ascbin=88tmaxaln-----10&seltype=2&
uid=C0G2199 at Figure 21 and Galperin MY et al. (2015) Nucleic Acids Res.
43(Database issue) D261-9; Ausmees N et al. (2001)Microbiot Lett. 204(1):163-167; Paul R
et al.
(2004) Genes Dev. 18(6):715-727; Chan C et al. (2004) Proc. Natl. Acad Sci.
U.S.A.
101(49):17084-17089; Ryjenkov DA et at. (2005)1 Bacteriol. 187(5):1792-1798;
Aldridge P et al. (1999) Mot Microbiot 1999 Apr; 32(2):379-391; Pei J et al.
(2001)Proteins 2001 42(2):210-216; Tal Re! at. (1998)1 Bacteriol. 180(17):4416-4425; Marcher-Bauer et at.

-36-' W02017/049127 (2015) Nucleic Acids Res. 43(Database issue):D222-6). For example, exemplary cyclic di-nucleotide synthetase enzymes nucleic acid and amino acid sequences derived from publicly available sequence databases are provided below.
Table 1: DGC nucleotide and amino acid sequences SEQ ID NO: 1 Vibrio cholerae 01 str. C6706 Contig 56 DNA Sequence (GI:446210820 REGION 98731..100614) tcacgcaaag tgatgcattt ccatggcggt gagtactgat atttggttgc gtcccgatgt tttggattca tataaagcca gatcggctct tttgtagctt tggtcgggtg atgtgcaaac atcggtaaca ccaccactta gggtcacttg ttgatggtgt aatgaagcga tatgcaggcg tacgcggtta agtacttgtt cggcttcttc aatggaagtg taggggaaaa taatggcaaa ctcttctccg ccaatccgtg cgataaaatc cgattcccgt aactgatctt ggatgccttt cgcaacggtc cgtaacacta ggtccccttc gttgtgtccg aatttgtcgt taatgcgttt aaagtggtcg atatcaatga tagcaaggca gctctqggct tgatcgggat aacggcgacg cttagcgcac tctaaagaga tggtttgatc gaatttacgt cgattccaca aatcggttaa cgcatctttt tcgctcagct cacgcaggcg attctccagc gccttgcgat gtgaaatatc cacaaaagag gcaacgtaga attgaatgac attgtcttca tcgcggatgc tttgaatacg gagaatttcg gtgatgcttt cgccatcttt gcgtttgttg atcacttcac cttcccatac gccattgtct tgcagagctt tccacatctg catatagaat tcgactttgt gtaatccaga agcaaaaatg gacggctgct tacctttgac atcttcaaaa gtgtaaccac ttaggcgggt aaattcgttg tttactttga tgatgcgatt ctggcggtcg gtaatgacca ccgctgacat gccatccatc gctgctcgag ccaatttact gtcaaggcta ttttttaaat ggttgatgtt ccatgccgca aatccagccg caatgataga gagtagcgat aacactgtca ccgcttgact catcagtgcc cagcgcgcat ttgcgtaggt cttatctatt tctgccttat tgatgcgcag taccaatacc aaaggtttaa agtcaggtaa gacagaactg agatccactt tgatatagct aaaccaggtt tgattggata gagcaaagcc ttgttggttg agttggattt tttgccaaag ctctgggtgt tgggctgaaa agtggagtga agaggttgaa cgtgtaccgg atggcttgtg ttcactgagc agtaattctc ctgccgaatt caaaatatcc ggtgaatcaa actgatcata aataaaagag agacgttgat agagagactg tagcttcacc gtcacgacaa gaaaaccttg ccgttggcct tgatgctcaa tacccgtcac aaaacgaaag gtcggcagca taccagaagg cgtatctgct gacatcgcga cttgcgttgc ccaaacttga ggcgtcgtga gttgggcgta ttgagccaca atttgctggc tgaacggatc tgtcgtttga gcagattcaa caaaggtgac ttggtgccca tcgtaaatcg ctttaagttg ttcttttcct tgtctatcca gcaatctgaa tgaagagaaa atcgcttgcg atcttaacgt cacatcccac aatgttttga gttgactgag tgcttctttg cttggtgtgg tgacagccgt gaataaaagg tcatttttag ctaacagctg ggtggcttgg tgtgtgcttt ccagcattcg taacaagtca tgctgactga actcaagctg taagcgagtc tgtttttcaa cgctgctgac cgcttgagtc tcaagctggc tagcagcatg tatgaaatac agtgtaggaa tgaaaccaag tacaaacgca acaatggcaa attgtatgaa atatttacgg gctgaggtgt acat SEQ ID NO: 2 Vibrio cholerae 01 str. C6706 Contig 56 amino acid Sequence (WP_000288675.1)

- 37 -SEQ ID NO: 3 Vibrio cholerae 01 str. C6706 Contig 56 DNA Sequence (GI:446272186 REGION 240951..242336) ttatgaccag gtacgaaaga caacctggtt ctttccattc cgctttcctt cgtacattaa gctatcggca tcgtgcaaac tgaatggccg agctgggtgt aggtaaaatg cacagcctag gctgatggtg agtgacagag agtgttgggc attcactacc cacttttttt ctgcaactcg ttggcaaatt cgctcagcta actgctgcga ctcttctgca tttttaccac gcgctacaat agcaaactct tcaccaccaa tccttgcaaa gtaggtatcc gatgctaaag cttgtcgcac acacccaacc acgaaacaga tggcattatc tcctgcgcca tgcccaaagc gatcgttaat ggttttgaag tcatcaatat caaaaaccat caacgttaag ctgcctgatc gtgtttgttc cgcttcaaga tgttcaaaaa acgaacggcg attagcaatg cccgtcaagc tatccgtttt cgctaaatag gagagttttt gattggcttc ttcaagttgc tgtgttcgca atcgaacggt acgccgtagc tgaagagtat aaataacgat actgagtaag agacctgaag cgagaatcgg cattaagtaa cgtggataaa tcgtttcaat atgaacccat cgacttaaaa tacggttttt ctcattgcta cttaattgtg caaacccctg ctctacttgc tctaataaat ccctattgcc tttggcgacc gctggacgta attcctctga ataaagaaac ttcactggcg taaaatcttt cgcgccattg gaaaccacta tatagaaatt ggcgacctga gtatcggcca caaaaccatc taattctcgt cgctttgctg cagacatcat caattcattg ttggcgtact caatcaactt aagttgagga tattctcgtt gcatgaactc ttgttcaaat ccccctttta ctacacctaa tgagacgtta atggcccccg atagcagcgt atccaattta tcgcccaata acgtgcggtg tacgtagagt tgtgtatcga ttgtcagtaa aggttctgca aaatcgagat acgctaatct tgaagcagaa cggatcaaac cagcttgaac atcggatttg ccaagcttca ccgcttctag ggaatcattc caatccatca gttggaattc aatatcgaca tgattcgctt caccaaaagc caaccaaaaa tcaatcaata tgccagaagg ctgtccctgt tcatccaaat aagaataggg tttccatgct tttgagttgg caatagtcaa ggtttggcgc tctacagcct cactcattga tccgaataaa agcggccaag caatcatgag aagcagaaac agtttggtcg aaaagcgatg atccat SEQ ID NO: 4 Vibrio cholerae 01 str. C6706 Contig 56 amino acid Sequence (WP 000350041.1) SEQ ID NO: 5 Vibrio cholerae 01 str. C6706 Contig 20 DNA Sequence (G1:446493741 REGION 153278..154204) atgatagaac ttaatagaat tgaagagctt tttgataacc aacagttctc cttgcacgaa ctcgtgttga acgaactggg agtctatgtc ttcgtcaaaa atcgccgcgg cgagtatctc tatgctaacc ctctgactct aaagttgttt gaagcggatg cacaatcgtt gtttggcaag accgatcaug atttttttca tgatgatcaa ctcagtgata tcttggcggc cgatcaacag gtgtttgaaa ctcgtctctc ggttatccat gaagaacgag ccatcgccaa atccaatggt ttggttcgga tttatcgcgc agtcaaacac cctatcttgc accgagtgac aggcgaagtg attgggctga ttggagtttc aaccgatatc accgatatcg tggaactgcg tgagcagcta tatcagctcg ccaataccga ttctttaact cagctgtgta atcggcgtaa attgtgggcc gattttcgcg ccgccttcgc tcgcgcaaaa cgtttaagac agccgttaag ttgcatctct atcgatattg ataatttcaa actgatcaat gaccaatttg gtcacgataa aggtgatgaa gtcctgtgtt ttctcgccaa actatttcag agcgtcatct ctgaccatca tttttgtggt cgtgtgggag gtgaagagtt catcatcgtt ttggaaaata cgcacgtaga gacggctttt catttggctg aacagatccg ccaacgtttt gcagagcatc cgttctttga acaaaacgag cacatctacc tctgtgcggg ggtttccagc ttgcatcatg gtgatcatga cattgccgat

-38-atttatcgac gctccgatca agcactgtat aaagccaagc gtaatggtcg taaccgttgc tgtatctatc gccaatccac agaataa SEO ID NO: 6 Vibrio cholerae 01 str. C6706 Contig 20 amino acid Sequence (WP 000571595.1) SEQ ID NO: 7 Vibrio cholerae 01 str. C6706 Contik_20 DNA Sequence (QI:446446879 REGION 171467..172840) tcaaaagcga tagagtgggt tttgcctacg cttagcggta tacatacgtt catcggccag tttgaacatt tcatcaggtg tggcaaacga ctggtcatac aaagcatatc cgatacttac acgaacatgg ataagcttgt cgtcataaac gatgggcgtt tcagaaatcc tttttaaaat attgtcactg actttaagca cgtcttgttc acgatgaatt cgtggaatta acacgagaaa ctcatccccc ccaatccgcg ccaccagatc ggaaacccgc aggctcgatt taattctttc cgcacaagcc accagcactt tatcgcctgc gctatgtcca tgggaatcgt tgatagattt aaaacggtca atatcaatgt tcaacaaagc aaagttacct tcgctatgag agcgcttagc attttcaaag tagtgttcaa tggtatagat aaaatagcgc cgattcggca agtgggttaa agggtcatgt agcgcacgct cctccgcgac ttgataaagg cgcatgataa cgccaaagcc tgccatcaat accaataaca ccgagtatcc caacaagcgc actgcatttc gggtatacca agataactgc tgtagtaaat cttgcttttc agcgaccgca attcgccaac ttccgtaagg gaaatagaca ttctcttgtg caaaagcgtg ctcaaatact cgaggctctc caaaaaacac gtccccctca ctgccacggc tgtctaaacc acgaatcgca acctgaaaat gctccccaaa gctgtaaata ctggttgctg aaagcaatga atcccaatcc atcaccacac tcagtacccc ccaataacgc gtatccttcg gtgggtcgta gaatatcggt tctcgaatca ccagcgcgcg cccaccttga acgagatcga caggtccaga gacgaacgtc tgtttgattt cacgtgcttt ttttattgac tgccactgct gaggaacggt gcggtaatcc aaaccgagta gtgcattggt ttgaggaagc ggatagctga aagcgaccac atcattaggg gcgataccta atgagcgtaa gtgatcgcta ttcctgatca ccgccgctga aagcggctcc cattgataga tattgaggtc gggatctagg gttaacaggg ttgttaaacc ttttacggta tagatatcac ccaaaatctc agcttctaat tgaaaacgta cgatggaaag atcttcttta gcttgttgac gtaaaccctc ttgtagatca cgtgtatggc taatatgaag ggattcaata accgcaatgc ccaaaaagag taaggcgaga aaataaattg agacatactt atatttgtgc gaggttaacc ccat SEO ID NO: 8 Vibrio cholerae 01 str. C6706 Contig 20 amino acid Sequence (WP 000524734.1) SEQ ID NO: 9 Vibrio cholerae 01 str. C6706 Contig 20 DNA Sequence (GI:446446879 REGION 171467..172840) tcaaaagcga tagagtgggt tttgcctacg cttagcggta tacatacgtt catcggccag tttgaacatt tcatcaggtg tggcaaacga ctggtcatac aaagcatatc cgatacttac

- 39 -acgaacatgg ataagcttgt cgtcataaac gatgggcgtt tcagaaatcc tttttaaaat attgtcactg actttaagca cgtcttgttc acgatgaatt cgtggaatta acacgagaaa ctcatccccc ccaatccgcg ccaccagatc ggaaacccgc aggctcgatt taattctttc cgcacaagcc accagcactt tatcgcctgc gctatgtcca tgggaatcgt tgatagattt aaaacggtca atatcaatgt tcaacaaagc aaagttacct tcgctatgag agcgcttagc attttcaaag tagtgttcaa tggtatagat aaaatagcgc cgattcggca agtgggttaa agggtcatgt agcgcacgct cctccgcgac ttgataaagg cgcatgataa cgccaaagcc tgccatcaat accaataaca ccgagtatcc caacaagcgc actgcatttc gggtatacca agataactgc tgtagtaaat cttgcttttc agcgaccgca attcgccaac ttccgtaagg gaaatagaca ttctcttgtg caaaagcgtg ctcaaatact cgaggctctc caaaaaacac gtccccctca ctgccacggc tgtctaaacc acgaatcgca acctgaaaat gctccccaaa gctgtaaata ctggttgctg aaagcaatga atcccaatcc atcaccacac tcagtacccc ccaataacgc gtatccttcg gtgggtcgta gaatatcggt tctcgaatca ccagcgcgcg cccaccttga acgagatcga caggtccaga gacgaacgtc tgtttgattt cacgtgcttt ttttattgac tgccactgct gaggaacggt gcggtaatcc aaaccgagta gtgcattggt ttgaggaagc ggatagctga aagcgaccac atcattaggg gcgataccta atgagcgtaa gtgatcgcta ttcctgatca ccgccgctga aagcggctcc cattgataga tattgaggtc gggatctagg gttaacaggg ttgttaaacc ttttacggta tagatatcac ccaaaatctc agcttctaat tgaaaacgta cgatggaaag atcttcttta gcttgttgac gtaaaccctc ttgtagatca cgtgtatggc taatatgaag ggattcaata accgcaatgc ccaaaaagag taaggcgaga aaataaattg agacatactt atatttgtgc gaggttaacc ccat SEQ ID NO: 10 Vibrio cholerae 01 str. C6706 Contig 20 amino acid Sequence (WP 000524734.1) SEQ ID NO: 11 Vibrio cholerae 01 str. C6706 Conti g 20 DNA Sequence (G1:446298852 REGION 177406..178581) atggatagct ttgctggcaa ccaattaaaa gagatgacag agatgcgttt tgctcgtaag cagcatattg tcctgatcag ctctggtgtt gctaccgcta tttttcttgg gtttgccctt tactactatt ttaaccatca acccctgtca tccggtttat tgttattaag cggtattgtc accttattga atatgatttc gctgaatcgt caccgcgaat tacacactca agccgattta attctgtcat taattctgct cacttatgcg ctggccttag tcagcaatgc tcagcatgaa ttatcgcatc tcttatggtt atatccgctc atcaccactt tagtcatgat taaccctttt cggttaggct tggtttacag tgcagcgata tgcttagcga tgaccgcctc tatccttttt aatccggcac aaactggctc gtaccctatt gcacagacct attttttagt aagtctattt acgctgacga ttatctgtaa taccgcttct ttctttttct caaaagcgat caattatatt cataccctat accaagaagg tattgaagag ttggcttatc ttgatccgtt aacgggctta gccaatcgtt ggagctttga aacttgggcc acagaaaagc tcaaagaaca acagagttcg aataccatta ccgcgcttgt ttttctggat attgataatt tcaaacgcat taatgacagt tacggccatg atgttggcga tcaggtgtta aaacattttg cacaccgtct acgcaataat attcgtaata aagatcgagc caccaatcaa catgattatt ccattgctcg atttgctggt gatgagtttg tgctcttgtt atatggtgtg cgaaatttgc gtgatctcga taatattctc aaccgtatct gtaatctctt cgtcgaccgc tatcctgaga cggatatgct caacaacctc acggtgagta taggggcagc tatttatccc aaagatgcga tcactctgcc ggaactaacc cgctgcgcag ataaagccat gtatgccgct aaacacggtg gaaaaaatca gtaccgctat taccatgatg ccgctttccc tccggctgta gaaaccgtat taggcagtca gcccgttgag gctcctaacg taactccact gaaaaaagcg cactaa

-40 -SEQ ID NO: 12 Vibrio cholerae 01 str. C6706 Contig 20 amino acid Sequence (WP 000376707.1) SEQ ID NO: 13 Vibrio cholerae 01 str. C6706 Contig 30 DNA Sequence (GI:446803291 REGION 173493..173939) atgctagcgt tacctgcgga gtttgagcaa ttccattgga tggtcgatat ggttcagaat gtcgatatgg gattgattgt gattaaccga gactacaacg tgcaagtgtg gaatgggttt atgacccatc atagcggtaa gcaagctcat gatgttattg gtaaatctct gttcgagatt tttccagaga tccctgtgga gtggtttaag ttaaaaacca aaccggtgta cgatctgggt tgccgtagtt ttattacttg gcagcagcgc ccttatttgt tccattgccg taatgtgcgc ccagtgactc agcaagccaa atttatgtat caaaacgtca cgcttaaccc aatgcgtaca ccgacaggcg cgataaattc actcttctta tccattcaag atgcaacaag tgaagccctt gtttctcaac aagcttcttc tcaataa SEO ID NO: 14 Vibrio cholerae 01 str. C6706 Contig 30 amino acid Sequence (WP 000880547.1) SE0 ID NO: 15 Vibrio cholerae 01 str. C6706 Contig 42 DNA Sequence (GI:446975354 REGION 107290..108807) ttagacaaaa tttcgcacaa cgtatcgatc tcgtccgtgt tctttcgcat gataaagtgc catatccgcc tgatggaaca aagagagata agactccatc tttggagaaa tagcatacac accaccaatg ctcaccgtta gatattggca gagtgcatca accggatttg caatcgcgag ctgctcgatt ttgcttctca tctgttgtgc atactgttct gcatcaaatg cacagtccga agctaaaaca acacaaaact cttctccccc aaagcgcgcc acgattttct cgccatggaa ctccaccgat tggagcacat cagcaacgga acataaggct tcatcgccag ccaaatgacc aaagctgtca ttgaaacgtt tgaaaaaatc gatatcgaca agaaacagca ccagataggc ttgcggacga tcgctcaaat aacttttaag ctgcttttct aaatggcgac gattggaaat gcgggttagt ggatcatgct cagactgcca acgtaacact tgttgactat cctccaattg tccgacgatt cggttgatcg tagtggcaaa ttctttcatc tccgatgaga taaaagtact cgcatccggc atttttccgc ccgatgtttt aaattgttgc aacacttgac tggcggtcgt gatcggtttg atcaaggcaa tcaccaccca taaattgact aagtacatca ccagtgaaaa gaacagcaaa gcaagaattt cttcggttcg aatgaaggga ggatgcttaa tgtgatggtt aattttaaac aacacactgg aattaccgct gtaatcgagt tgcttgatgt atgaaacatc cacttcgtct tgcggtaagg gcgcatcatt tttacaggtt aagacttcaa tatcgacacc agtggcttgc tcaaccacat tcgcaaactg ggcgcggact tttttaataa agattaagaa acctttgtta caccctttcc catcactgtc acagacacga gccgtggcag ctaaataggg ctcatcctcc accaccatat aacgaacgga agtcgagatt tcatccacac ttaaacgtgt cgcctgctgt aaaatacgtg aaaaatccgg caataagtgc tcatagctag agctctgccc cgttgctgcg tcatatttct tgccccaaac caaattgccc tcaggatcat agataaatac gccatcgagg aattgtgaac tgaaagcgtg ctctccaata ttgctttgtg tgaactcaag ggtgggtttt gcaatgaagt ctgccatttc atcccaagcg gcataatctg ccaaagaagc ccccatcgcc ttacgttcta acgacaacaa ggtttcaacc cgctgcaact cggcctgttg taactgcagc acttgcgcaa cttcacgatc atgtgaccag aaatatttaa aggtcagata

-41-aaacattaaa aagcctaaca ccaccgctaa cgcattgagt gtcgttagcc agcgtaggct aaagttattt aaattcat SE0 ID NO: 16 Vibrio cholerae 01 str. C6706 Contig 42 amino acid Sequence (WP 001052610.1) 181 KGCNKGFLIF IKKVRAQFAN VVEQATgVDI EVLTCKNDAP LPQDEVDVSY IKQLDYSGNS

SE0 ID NO: 17 Vibrio cholerae 01 str. C6706 Contig 42 DNA Sequence (G1:447036588 REGION 195345..197084) ttagtggttt ggttgataaa ttgaggtctg attgcggcca ttcgctttgg cttggtataa agcccgatcc gctagctcaa cCatttgctc aggtacatcc tcaggccgag gaataagcgt cactatgcct aagctgacgg taatcctatc ggcaacctta gaatgatcat gtggaatcgc taatccacga actttctcat ggattcgctc tgcgaccagt attgctccgg actgtggtgt attgggcagc aaaataccaa actcttctcc cccgtagcgg gcaacacaat cagaatggcg attggcgact tgagtaaagg caatcgctat ctgtttgagc gtctcatcgc ccatcaaatg gccataagcg tcgttgtaat ctttgaaata atcgacatca cacagaatga tgcttaatgg tttgccttca cgcacatgca aatgccagag ggtatgcagt tgttcatcaa aacgacgacg attggcaaca tgagtcaagc tatctaaaaa gcttaggcgt tccagctctt ggttggcggc ttctaattgt tcagcggcga gatagcgctc cgacacatct cgcgccatga tcagcacgcc attggtgccc gaagccggat ctcgaaaagg cgatttcaca acatcaaacc agataaactc accatctgag cgttcaattc tgtcgatgta gcgcagagac ttaccttggt gcaggacttg gctatccgta tcggaaagac gcgcatagat gtgctcgggg atcacatctt gcagccgttt accaaccaga tctgacactt ccgcgatccc gagagcttcc acaaacggct ggttacaggc ttggtagacc atgttttcat tgaagatacc aatcgaatcg gggctagatt ctaagatgtt ttgtaaaatc gtatcgcgct gtgccaatgc cacttcggtg tcacggcgtt tttccatctc ttctcttaat tgacgctgca tgttgtacca gtcggtcaca tcatgactga tgccaagtag cccaatattt tcaccttgcg gcgacatcaa tacccgttgg taggtttcta acagacagct gcgcccatca ggcgtcacag tccagcaacg ctgactcgtg cgccctttca taatgccttt aaaagtagcg ctgccctctt caatccggcc ttgccaaaac tgatcaaacg ctcggttggt tgcgattaag tggccttcgg tacttttaat aaaaatcagc tcggagaggg aatCaagtgc cgtgcgcgct atcgccagtg agtggcgctc ttgttgaatg tcatggctgg gacactcaaa accaatcaca ttcactagcc ataatttctt cggccaacga cgtaagagcg aagctgagat ctctagagtt tgggtcaaat tgcccggcac aggccaaagc agagggaCgg aacgcttttg ctgtgcactg ctggcgagcg ctcgataaaa agcttgctga ctctcttcac tctgctcggc agaaaacaga tagtgacgtc ccaccaagcg gatccccagt aacaaatacg cggcaagatt ggcacgtaaa acgcgatcct ctcctaccaa gagcatccct gacggtgcat ggtgaagtaa ctgaatccac tgttgaggtt gaacatagcg ctgccatcct gaaaaaagcc ataacccacc accaagcaca agcccggCag cgaacaagaa acgtacaaat tcagagagaa attcaggcat SEQ ID NO: 18 Vibrio cholerae 01 str. C6706 Contig 42 amino acid Sequence (WP 001113844.1)

- 42 -' W02017/049127 SEQ ID NO: 19 Vibrio cholerae 01 str. C6706 Contig 40 DNA Sequence (GI:446834936 REGION 93475..95058) ttacataaag tcgaacatcc tacctgaatt gaaggcataa ttcgattcta ccttgctgca ttgctgcgca atcgatacac gatttcgacc tttcgattta ctgagataga gctgatcatc aacactctgt aaaatttccg gctcactgta ctcacagtta atgctcgccc caatactgat ggttaaggtt aatggtgtct cggcattgag catcacaggt tctgcttcga ccactttacg gatccgctct agataagtat aaagcgccgt ttcatcagta acggatgaca agatggcaaa ctcatcaccg ccgaaacggg caaaaatatc cgattcaacc aactcttttt tgaccacatc aaccacatgc gttaaagcgt aatcccccgc taaatgccca tagctgtcgt tgatttgctt aaagcggtcg atatcaaatg aaatcaaggt aaaggattgt ttttcatcta acattttgca caaatgctga ctaaagaagc ggcggttata gatgttggtc aaactgtcat gctccaccag ataacgcagc tctgcggtac gctcctcaat catatctgtc agccgttgtt tctcttccag ttgcattcgc atgatgtagc taagcagcag agaaataata acgccaccca accctaagcc cagtagcacc cactcttcac tatggttaat cggctgatgc agttcaaact ccagcaccca atcacggttt ggcaacacca atttgcgctc tattttgggt tcatcatccg ctcgccacat cgggctttga taaagaaccg gactgtcttc cgaatcaaat ccggtgtcaa tcacgcgcat atcgagatct tgttccatga cgctgatttg gaccagtttc tcgaaatagg tggataggcg caccaccccg accatcacac caagtaagct gcgatcatct tctgaagaaa aaacagggtg atagaccaac atqccatctt tgacgatcga cttatcaatc ccatcttgta gcaggcgcac tttatccgaa acattcggcc gacgattaac gacaatatcc gccagtattc gtttgaaacg ttcacgctcc gagtaaaagc ctaacagttt acgattgtca taattgagtg gataaatatc cgataaaacg tatttcgctt ggtcatccgt accgaaaccg tatttgatct ctcccgtttt tggcaccgtg tacaaagtga actcaggaaa acgttgctgc attcgcgcgg taaaagtttc agcctgaggc ggctcaactt tcactaacca ttgtaaagca atcaggcttt gtgaaccttt aagagtctct tctgcgaaag tgtgaaaacg cacccagtca tcgcttgtgc ttgagcggaa aaagttggcg gcagagccga taaaatggat atcaccatcg acaaactgtt gcagtgccat agtttgccta tccgcaaggt tttccagcag agtacgatta tggcgcagct gtaatgagta tgcggtgtaa accacaaaca cagtcagaag cagagaaaac aacagtacca gcaagggcac aatcacgcgc acatgtttga gcat SE0 ID NO: 20 Vibrio cholerae 01 str. C6706 Contig 40 amino acid Sequence (WP 000912192.1) SEQ ID NO: 21 Vibrio cholerae 01 str. C6706 Contig 40 DNA Sequence (G1:446533459 REGION 103406..104737) tcagctcact aaactggtgt gatcgtgctt atcttggtgg gcgcaataca ccgtattgcc ggattgatgt tttgcggtgt acatcgcctc atcagcaata cgcaataatt caggtacttg ggtcgcttgc tctggatata aggcgacacc aatactcacc ccaatctcta agctctcttg gttaagttga agcggctttt gtagtttttc tagcatctga taagccttat tgataacgcc actgtgatcc tgcagcagat ctaggcatac cacaaattca tcccccccta agcgcccaca aaaatccgat tctcgtatcg accctttgag ccgttgagcg atttcttgca agacaagatc

- 43 -, W02017/049127 acctacttcg tgccctttgg tgtcattgat ttctttaaat ttatctaagt caaaaaacag caaagccagc ttcatgtttg agcgcttcgc tttaattaac gcgtgactaa gctgctgttt aaaggcacgg cggttcaaaa tacctgtcaa tgaatctctt tctgacaaga aacgtaattc cgctttttga cgctctaatt tggcggtttt tcttgccact tccgcttgta actcatcttt ggtaacggtt gtgctttgca gcgaagcctt catttgattg aaaaactgag ttaattgaac aaactcttgt tcattatttt gagtggaaat tcggctggcg agatcccctt tcgccatttg ttcaatccct tcttggagag ttttacatcc atgtcggaag cggcgtaaca ccaccagcgc gataccacag acaaccgatg agaagagcag taagtgcgcc atggtggtta acaataaata gcgttgatta ttaatgctct cttccatgac ttgacgctga aaataggcca actcctcatt catgttttgc accaaaatat tgtatcgaga gtgaagtagc tcataagttc cgatgccatc gaccaactta gtaatgcccg attcttcggc catgtagcgc tcttgttcta atagcccggc taaactgtta ttcattcttt ggatgccggc taagtgttgc ccaaagaccg tttccatctc gagctgccca gccaaaacct gctgcgcacg ataaacctgc tctaagctat gagcatcgtt gtattgcaga aagacccaga gctggctacg caacatggca atgctgtttt ggatttccaa aatcgtatcc agctcagcat tggtttgctg ctgccgctga tctaagttca gtaatgagaa agcaataaaa ccaactaaca gcagtgatgc aataaacagt aacgtcattt tgcggtttaa tgagttgatc aa SE0 ID NO: 22 Vibrio cholerae 01 str. C6706 Contig 40 amino acid Sequence (WP 000610805.1) SEG ID NO: 23 Vibrio cholerae 01 str. C6706 Contig 37 DNA Seouence (01:446848493 REGION 64235..66256) atgctactta acgctttttc acgccgagtc ttcctttggc taggttggct attgatttcc accagcagtt tagccgctac atctacgacg tataaggtcg ccaccgaagc ggatgacgtg gtgactcgtg tgctttttga ttcgattgct caccacttca accttgaaat tgaatacgtc aactacccca gttttaacga tattctggtg gcgatagaga ctggcaacgc cgattttgct gccaacatta cttacactga tttgcgtgct caacgttttg atttttcaag accaaccaac atcgagtaca cctatctcta cagttatggt ggcctacgtt tacccgagtt gcgcctcgtg ggtatcccga aaggaaccac ctacgggacc ctactaaaag aacactatcc ctatatccag caagttgagt atgaagggca tttagaagcg ctcactttgc tggaaagtgg ccgagtagac ggagtggttg atgcgatcaa tcagctcaaa cctatgctac tgaaagggct tgatgtacaa ctccttaacg accaattacc gattcagcct gtttctattg tgacgcctaa aggcaaacac tcagcgctat tgggcaagat tgaaaaatac gcgcattcgg ctcacgtaca acgtttattg cgtgaatcga tccaaaagta tcaattggac atccgtaagc aagctctgcg tcaatccgtg gttgagagcg gactcaacgt gcagcgtgta ttgcgtgtta agctagagaa caacccgcaa tatgcacttt atcagccaga cggttcggtt cgtgggatca gtgctgatgt tgtgtttcag gcctgtgaga tgctactgct gaaatgcgaa ttggtcagta atggtcaaga aacatgggag agcatgtttg atgatttaca ggataaaagc atcgatattt tggctcctat aacggtttct cagcagcgta aaaacctcgc ttacttcagt gaaagctact accacccaca agcgattttg gtcaaacgtg aacactataa agacgatgtg tatagcaatg tgtctgagtt ggtggctgaa cgtattggcg tcatcaaaga cgattttttt gaagagctgt tacagcagat gctgccgaac aagatcttgt tcagctacgc aagtcaggaa gagaaagttc aagccttact gaataaagag gtggactaca tagtgctcaa tagagccaat tttaatctct tgcttcgcga gtcaacggag atgttaccga ttgtagaaga caccatgatt ggcagtttct accaatatga cattgcgata ggttttgcta aaaatccact tggtgcaact ctggcacctc ttttctctcg ggcaattaaa atgctcaata ccgaacagat catacatacc tatgattatc agccaaattg gcgagccaca ttacttgcgg aaaagaaata tcagcgcagt actcaatggc tttttgccat ggctttcatc gttttgttta tggtggcgtt ttacctccat ggcatatcac ataccgataa ccttactaag

- 44 -ttgcgcaatc gtcgcgcttt gtataaccga taccgccgcg ggttatcgcc tcgcctaagc ttggtttatc ttgacgtgaa tacgtttaaa tcaatcaacg atcagtatgg acatgaagtg ggtgacaaag tccttaagca gttggctcag cgcatcgaag cggtatggcg tgggcgcagc tatcggattg gtggggatga atttatttta atcggtgaat gttctgctaa gcggcttgaa catgtggttg cgcaatgtga acgttttatg tttgtggatg cagagcgcga tgtcagtttt gaagtgagtg tggcgattgg tattgctaag aatcgtgagc ggaccgaatc actcaatgag gtgatgcacc aagcggatat tgcgatgtat cgcgctaagg cggaatcgac gcaatcgcca tttcaggctg ccagcaaggt aaaaggatta cacatcgttt aa SEO ID NO: 24 Vibrio cholerae 01 str. C6706 Contig 37 amino acid Sequende (WP 000925749.1) SEO ID NO: 25 Vibrio cholerae 01 str. C6706 Contig 36 DNA Sequence (GI:446054248 REGION 42225..43517) ctatctgaac tgatcctgct tgagttcttt cgcactggga agaggcagga tctcttcccc cattcgataa atatgatagc catgtttgcc tctgtatttg acccagtaca tggctttatc ggcttgtagc agcagttttt ctaagtcaat gtgcagactg ttcatatgac taatcccgat actgcaaccc acttgcgcac tctgctgacc caatccaatc ggctcagagg aggattcgat caactgagcc gcaaaccgct cgatagattc ggcaacaaat tcatccagcg gaatgtaaat agcaaactca tcaccaccga gccgtccgac cacaaaatca gaaaaatgtg tttgcgccaa ggcataaaaa cgtttggcga tttcacgtaa tacctcatcg ccagccgcat gccccaaggt atcattcacc tgcttaaaac catccaaatc aatcaatagc agcaccatag tggtgctagc acgctgtttg cggagcacga acttctcaca acctaaacgg ttttttagtc ccgtcagcgt gtcttgttcg gcaatggtgc gatagtagct ttcccaacgt tcaatctgtt gacgcagctc gcgttcacgc agtaaagctt gatgggaagc gtcgataaat tcattgatgc ttttggccac caaaccaatc tcgttgtggt gatcttctgc ggctaccgcc actttgcgat catgatctgg ccgcacttcg gataacgcct gtgaaagatc cgtcaggggt ttaccgacca agcggcgaac gatccagata agcgcaataa aagtcacgag aaactggatc aaaaccacgg ctatctgatc aagaatctga ttaatggctt gctgacgaat cacctgatga tcctcatgaa tcatcagata gccaatcaaa ttaccatcta cgggagaatc taatcggtag cggttcgcat cactccaata attctgctct ttgtaggttg aggggatggt ggtgcgctca aagacaatgc catccacgct ggctaactta accgcattga tctcttgatg aagcagcaac gcatccatca cctcggaggc aatatcgtaa ttattcacat acagtgcaat ggccgccgag ttactcaagg agagcgcaag cttctcttcc agctcttgtt tttgctgctc aacactctgt atgccgcgcg gaataatgat ggccaaaatg atcagcaaat acccaagtgc acacagtgaa atcatcttca gcaagcgatt aaccagtggc gaagttcgcg tttgatcagt cat SEO ID NO: 26 Vibrio cholerae 01 str. C6706 Contig 36 amino acid Sequence (WP 000132103.1) 181 TDLSQALSEV RPDHDRKVAV AAEDHHNEIG LVAKSINEFI aASHQALLRE RELRQQIERW

- 45 -SEQ ID NO: 27 Vibrio cholerae 01 str. C6706 Contig 62 DNA Sequence (GI:480994257 REGION 1..1003) agcgcatacg ctcaagtagg gcttgctcac gttgctccgc taagagtaag cgttcagaaa gtgaagacat ctcgcgcagt aaaggcgcca ttttcagctt gagctgttct agctccgtct gttctttgag cgcggtctgg ctacgagcca ctaaactgct cagctcgcca ttcatctctt ggcggtgtgc catgtaactc tggctttgct caagattttg agtcgcgctt tttaggttat tgccaatcga gagattcact tgctcgagaa aggcttcggc tgctttgcgc tcagcatggc ttccatcgac gactaaacgc agtacttcaa gggtgagctc aagcagggta tgggtattga cgccaagcag aagcttggtt cggatatcgg tcagttgatc acccgattca ccattgaaat ccaactcagt aatcaagtgt tgtaaatcaa cggcaagtcg atgcagcagt tctcgatccg cttgttgagt aagctcattg agcgccaaat tgggattggc acattgaatt ttgaccgcgc gttcataaat ttccagcaaa cgcaaagctt gctgagtttt ttccagcggc tgtgcggcgc taaaactcag cagatctcga agatcgcgtt tgatcttggc gggtaagccg gggacgcgca gtagcgtttc accactgtgc tgtagctggc tatccagatg actcgtttgt ttgtccatgg ccaatgactg ttgtttcaac atgcgttcca gtacggctaa tttcgggatc agcgtactga tgtctttttg ttgttctaat gcaaaacaga gttcttctaa actttggttt agtcgagagc tactgccgcg gcaagtcgta gccaaggaag tgaccattcg tttaagaact tgctgctctc ggttaaattt gaacgaagta tccctttgtg tcaaacgtac ttgttctaac tgagatttca gtttttgaag ctctgcttgg atatcttgtt ctagaacgcc cat SEQ ID NO: 28 Vibrio cholerae 01 str. C6706 Contig 62 amino acid Sequence (WP 000538436.1) SEQ ID NO: 29 Vibrio cholerae 01 str. C6706 Contig 27 DNA Sequence (GI:480994257 REGION 1..563) atagcaaaga tcagatggaa gccctgtctg atttggcaca agattatcgt cgccgccttg aagatcaagc attgcgcgca caactcgatc ctctgaccaa agtgtacaac cgcagcagct ttactgagcg acttgaacat gagtatcgcc gctggatccg tacgcaacac aatttgcggg tagtgctgtt tgatattgat aaattcaaat cgatcaacga cagctttggc tacaccgcag gcgataaggc cttaagtatc attgctcgca ccatcaaaaa agaattacga gacagtgaca ccgtggctcg cttctctggt gaagagttca ttctgttact gcctgaacgc tccgataatg agagttacca gattattcac cagatccagc tcaacgtgtc gaaactaccg ttcaagttcc gcgataagag cctaaccatc acgctgtctg cggcgagtat ccgcttcatg gattcagata cccccgaaac ggttcttgat cgtttaaatc tgacgctaag tgaagccaaa catatcggtc caagtcagtt agtttggaaa taa SEQ ID NO: 30 Vibrio cholerae 01 str. C6706 Contig 27 amino acid Sequence (WP 001888804.11

- 46 -121 RTKLLLGvNT HTLLELTLEV LRLVVDGSHA ERKAAEAFLE QVNLSIGNNL KSATQNLEQS

SEQ ID NO: 31 Vibrio cholerae 01 biovar El Tor str. N16961 amino acid Sequence (NP 233340.1 GI:15601709) SEQ ID NO: 32 Vibrio cholerae 01 biovar El Tor str. N16961 DNA Sequence (D0776083.1 GI:109706432) 1 atgatgacaa ctgaagattt caaaaaatcc acggctaact taaaaaaagt cgtaccttta 61 atgatgaaac atcatgtcgc ggccaccccc gtgaactatg ccttgtggta tacctacgtc 121 gaccaagcca ttccgcaact gaatgcggaa atggactctg tattgaaaaa ttttgggctt 181 tgcccacccg cttctggtga acatctttac caacaataca ttgcgaccaa agcagaaacc 241 aatattaatc agttacgtgc gaatgttgag gtacttcttg gtgaaattag cagttcaatg 301 agtgatacgc tcagtgacac cagttccttt gctaatgtga ttgataaaag ctttaaggat 361 ttagagcgcg tcgagcaaga caatctctcg attgaagaag taatgacggt gatccgccgc 421 ttggtgagtg actctaaaga tattcgacac tcaaccaatt tcctaaataa tcaactgaac 481 gcggcaacac tagaaatctc tcgtcttaaa gagcagctgg cgaaagttca gaaagatgct 541 ctgtttgaca gtttatctgg actctataac cgccgagctt ttgatggcga tatgttcacg 601 ctgatccatg caggtcaaca agtcagcctg atcatgctcg acatcgacca cttcaaagcc 661 cttaatgata actatggcca cctgtttggt gaccaaatta tccgtgcgat cgccaaacgt 721 cttcaaagcc tatgccgtga cggcgtgaca gcttatcgtt atggcggtga agagtttgca 781 ctgattgctc cgcacaaatc gctgcgtatt gcacgccagt ttgctgaatc ggtgcgacgt 841 tcaatagaaa agctcaccgt aaaagatcgg cgtagcggtc aatcggtcgg tagcattacc 901 gcttcgtttg gtgtagtaga aaagattgaa ggtgactctt tggagtctct tatcggtcga 961 gcggatggat tgctgtatga agcgaaaaat ctgggccgca atcgagtcat gccgctcttg SEQ ID NO: 33 Vibrio cholerae VCA0956 01 biovar El Tor str. N16961 chromosome II DNA Sequence (Ri115600771:904820-905839, NC 002506.1) GTGATGACAACTGAAGATTTCAAAAAATCCACGGCTAACTTAAAAAAAGTCGTACCTTTAATGATGAAAC
ATCATGTCGCGGCCACCCCCGTGAACTATGCCTTGTGGTATACCTACGTCGACCAAGCCATTCCGCAACT
GAATGCGGAAATGGACTCTGTATTGAAAAATTTTGGGCTTTGCCCACCCGCTTCTGGTGAACATCTTTAC
CAACAATACATTGCGACCAAAGCAGAAACCAATATTAATCAGTTACGTGCGAATGTTGAGGTACTTCTTG
GT GAAATTAGCAGTT CAATGAGT GATACGCT CAGT GACACCAGTTCCTTT GCTAAT GT GAT T
GATAAAAG
CTTTAAGGATTTAGAGCGCGTCGAGCAAGACAAT CT CTCGATTGAAGAAGTAATGACGGTGAT CCGCCGC
TT GGTGAGTGACTCTAAAGATATTCGACACTCAACCAAT TTCCTAAATAATCAACTGAACGCGGCAACAC
TAGAAAT CTCT CGTCTTAAAGAGCAGCTGGCGAAAGTT CAGAAAGAT GCT CT GTTTGACAGTT TATCTGG
ACTCTATAACCGCCGAGCTTTTGATGGCGATATGTTCACGCTGATCCATGCAGGTCAACAAGTCAGCCTG
ATCATGCTCGACATCGACCACTTCAAAGCCCTTAATGATAACTATGGCCACCTGTTTGGTGACCAAATTA
TCCGTGCGATCGCCAAACGTCTTCAAAGCCTATGCCGTGACGGCGTGACAGCTTATCGTTATGGCGGTGA
AGAGTTTGCACTGATTGCTCCGCACAAATCGCTGCGTATTGCACGCCAGTTTGCTGAATCGGTGCGACGT
TCAATAGAAAAGCTCACCGTAAAAGATCGGCGTAGCGGTCAATCGGTCGGTAGCATTACCGCTTCGTTTG
GTGTAGTAGAAAAGATTGAAGGTGACTCTTTGGAGTCTCTTATCGGTCGAGCGGATGGATTGCTGTATGA
AGCGAAAAATCTGGGCCGCAATCGAGTCATGCCGCTCTAA

- 47 -SEQ ID NO: 34 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31434.1) SEO ID NO: 35 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (G1:695934235 REGION 195154..196539) atggatcatc gcttttcgac caaactgttt ctgcttctca tgattgcttg gccgctttta ttcggatcaa tgagtgaggc tgtagagcgc caaaccttga ctattgccaa ctcaaaagca tggaaaccct attcttattt ggatgaacag ggacagcctt ctggcatatt gattgatttt tggttggctt ttggtgaagc gaatcatgtc gatattgaat tccaactgat ggattggaat gattccctag aagcggtgaa gcttggcaaa tccgatgttc aagctggttt gatccgttct gcttcaagat tagcgtatct cgattttgca gaacctttac tgacaatcga tacacaactc tacgtacacc gcacgttatt gggcgataaa ttggatacgc tgctatcggg ggccattaac gtctcattag gtgtagtaaa agggggattt gaacaagagt tcatgcaacg agaatatcct caacttaagt tgattgagta cgccaacaat gaattgatga tgtctgcagc aaagcgacga gaattagatg gttttgtggc cgatactcag gtcgccaatt tctatatagt ggtttccaat ggcgcgaaag attttacgcc agtgaagttt ctttattcag aggaattacg tccagcggtc gccaaaggca atagggattt attagagcaa gtagagcagg ggtttgcaca attaagtagc aatgagaaaa accgtatttt aagtcgatgg gttcatattg aaacgattta tccacgttac ttaatgccga ttctcgcttc aggtctctta ctcagtatcg ttatttatac tcttcagcta cggcgtaccg ttcgattgcg aacacagcaa cttgaagaag ccaatcaaaa actctcctat ttagcgaaaa cggatagctt gacggacatt gctaatcgcc gttcgttttt tgaacatctt gaagcggaac aaacacgatc aggcagctta acgttgatgg tttttgatat tgatgacttc aaaaccatta acgatcgctt tgggcatggc gcaggagata atgccatctg tttcgtggtt gggtgtgtgc gacaagcttt agcatcggat acctactttg caaggattgg tggtgaagag tttgctattg tagcgcgtgg taaaaatgca gaagagtcgc agcagttagc tgagcgaatt tgccaacgag ttgcagaaaa aaagtgggta gtgaatgccc aacactctct gtcactcacc atcagcctag gctgtgcatt ttacctacac ccagctcggc cattcagttt gcacgatgcc gatagcttaa tgtacgaagg aaagcggaat ggaaagaacc aggttgtctt tcgtacctgg tcataa SEO ID NO: 36 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31434.1) 181 ELDGFVADTQ VANFYIVVSN aAKDFTPVKF LYSEELRPAV AKGNRDLLEQ VEQGFAQLSS

SEO ID NO: 37 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (G1:695934238 REGION 199457..200695) ttagctagcg actttgacac aattgcgccc agcttgcttc gctttataaa gtgccccatc cgctgctttg agtgcctcaa taggatggcg gtacagctca gaatcacaca cgccaatgct

- 48 -6-t7 4423.45322o 2522'532535 033235222D 3424633334 3222633E33 235E225355 4252223.532 2232233253 beubqbece2b bb12.46.3661 2232522354 3335222553 6425235423 233;422534 5222.323244 2554o-43353 44.4.4233353 3523522.4bb 2223454252 26343332ov 4455452243 oboo324.442 2632232223 5222332332 35.33.225233 5332533233 2335515536 2335323554 25.6325352o 5253435533 2223520253 3335232222 22.43423328 342328553p 3553544326 5335636442 33233.43332 3363423363 5232535536 2233625325 33236E6436 36136322235 323332.5223 2523282523 6522322042 3635332465 3324554333 3222334325 33023333E3 3335233332 5.535222332 324062413B B3=222332 2033233436 233 3322 3DqPP22223 brY4T4DBE,2 300P32POOD bEDqqqq020 3332333333 3283445323 235533323D 5223232254 5,23335338.4 42252.65235 534-4225444 324255332o T4253.43523 .4253234323 3333333336 322338433o 4336232435 22,54553253 5316443433 455223553e 2335522332 3526432.4M
5025454443 5344;3326o 3543642355 go-43=5324 2523E23454 2E3503522o .6322355,534 352233335o 253234022o 3353232234 355463322e 35233E12333 5333262325 32223.43533 322633E1334 332335220o 235563253e 333263622u -4332232252 2286682385 2425643534 25833:42344 343433426o 52235.34252 234.6326153 256635332o 2282333223 46.23132052 2522236823 3232=2335 3055323132 333433253e y8223352;3 533523=2o 3522332323 2362226533 532.25321.35 3302642352 335231.4525 343523233o 5133325232e 22254363.62 35234553E12 2333252643 3523352335 435323812o 4312353323 2433432334 #53332353 .4353.533643 23353.43223 2423444232 2236333523 433232454e (LI88.1769E NOIDa1 09E17E6569:ID) aouanbas yNa z wosocuattp 9L I z-laz oz apaa101P 0!"fili __ 6 :ON GI OHS
S Mal3AAONM9 NENS2AWIS5 VGIVISadlIVd H7A3V30qSI TZI7 SV ANADNHOVOM VMAqVDCIVVM qVaIdHEX'I2 SCOASISIII LAIGSMSMES 195 MR/SSE/TM RIONIGUNI SA621021'111 2IAcTrIA2IVI MONXIIRJ22 DSHAAVVMS TOE
NIMUSVA7 TIAGGOIGHS AIGNAMA3HG ATIWVqIIN21 SIHNIGOTIV 02iDdnIqd0 Ti7Z
N2VW2ADVSI I3WIISIAdA H3rIAAYNWSrl AISSAAAAGI TBT
VIDIFIVNIAV AAR-IT-121Sn dAA33I9aIrl SSHc1L2SIWI ONIMVNOI2r1 grlIVTASUS TZT
MS.41OVIV,W AdrIZOVAdT1 IS WIIIWL SNAIrldIME IOTIMAYTIN INVANVIHSO 19 MaIHVAITIVI rilgIAdrINAV 145rINSMINIA AN3rMqMIVA TIT1.1.49.33 MdS1A2SS3N
(1 -LEN Env) amanbas ppr ou!tuu z OLuosotuolip 9L I Z-1HZ I OZ u!vils avdapip oy(pA __ 8E :ON CII
OHS
.423852252 4358283224 6225356223 3225532285 3322225526 2422358352 3321355322 23352433.62 2313543832 3321283323 4233432523 3522333632 3353325445 6255662.454 2252522222 3328363623 3653223526 3.63222224.4 3544435431 325356323o 2335332335 3323223583 3522152332 23.42553142 25315225.43 1505522234 5532533225 5355420522 3445264223 2362343622 3522223552 323356232e 2222-455823 60552463.43 4323E23312 8388543534 32.55222526 3322452233 6838222422 3523226385 8228333582 3362335.663 3583536323 33.2333354o 4553225533 5E25366333 3343838882 8538238.465 3.634328858 53682-4.65.62 2338338838 835.62.3862.8 3853.545863 .5.4.332.42.835 8358486853 3238833135 3543864332 852.23.42388 3233228423 38253.42832 4823435383 3423532223 2358322854 2023562242 4222564663 2542322845 6224252333 5835823225 833222.4668 5322856353 3352323133 2335.422843 5336554.625 1E65344285 5233253353 3053628332 6333322234 3454242333 2503443228 4655253624 835.2633232 5036632285 3333432.2.83 4534235823 5333648338 4533333363 4838322333 3E83328355 253322.4828 4.533433334 3883353343 3333888235 5288538533 3356224533 5333423883 4385683534 3823383.88 5383363553 356363556 333.3.38538 86IZCO/9IOZSI1LIDd LZI6170/LIOZ
OM

caattctacg ttgcctcttt tgtggatatt tcacatcgca aggcgctgga gaatcgcctg cgtgagctga gcgaaaaaga tgcgttaacc gatttgtgga atcgacgtaa attcgatcaa accatctctt tagagtgcgc taagcgtcgc cgttatcccg atcaagccca gagctgcctt gctatcattg atatcgacca ctttaaacgc attaacgaca aattcggaca caacgaaggg gacctagtgt tacggaccgt tgcgaaaggc atccaagatc agttacggga atcggatttt atcgcacgga ttggcggaga agagtttgcc attattttcc cctacacttc cattgaagaa gccgaacaag tacttaaccg cgtacgcctg catatcgctt cattacacca tcaacaagtg accctaagtg gtggtgttac cgatgtttgc acatcacccg accaaagcta caaaagagcc gatctggctt tatatgaatc caaaacatcg ggacgcaacc aaatatcagt actcaccgcc atggaaatgc atcactttgc gtga SEO ID NO: 40 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31559.1) SEC) ID NO: 41 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (GI:695934436 REGION 430738..432621) atggcaccga tcctttcaca ctcgatcccg atcccttcta gcatgcaggc aaattggcag cagatgctca acctgctggc cgaagtgctg aaagtctcag ccaccctgat catgcgttta cgccatcacg atcttgatgt gttttgtacc agtgtcggca gtgacaatcc ataccaagtc ggcatgaccg aacgattagg cacaggcttg tattgtgaaa ctgtggtcaa tactcgccag atattgttag tcagtaacgc cgacctcgac ccattgtgga aggataaccc agatctggaa ttgggcatgc gcgcttactg tggcgtacca ttgcaatggc caaacggtga gctttttgga tctttgtgtg tcaccgatcg tcaagctcgc cagtttctta gtaccgatca gcaattgata aaaacctttg ctgaatcgat tgaagctcag cttaaaaccc tttaccaacg cgaaacgttg ttgcaaatga accaagattt gcacttcaaa gttcgtcata aaatgcaaag catcgcctcg ctgaaccaat ctctccatca agagatcgat aaacgccgtg ccgcagaaca gcagattgag tatcagcgca gtcacgacct tgggactggc tttctgaatc gcacggcatt ggagcagcag ctcgcgatgc agctggctca attggcggaa cacgaagagc tcgctgtgat tcatatcggt tttgccaatg cccgccaatt acaggcgcgg ctgggttacc acctttggga tgatgtgcta aagcagttac gtgagcgact tggtccggtg acggaggggg aattactgac cgctcgccct aactcgacca atttgacgct gatcttaaaa gcccatccgc tcgacaccca attaaatcag ctttgccatc gtttaattca cgctgggcaa gcgcaatttg tgacggaggg gctgcccgtt cacctcaacc cttatattgg tgtggccctt agccgtgaaa cacgcgatcc gcagcagcta ctgcgccatg ccgtcagcag catgttggcg tgtaaggact cgggatacaa agtgtttttt cactctcccg cattagccga taaccatgca cggcaaaatc aattggaaaa ctatttactg caagcggtgc gcaacaacga tctgctgctc tacttccaac ctaaagtcag catgaaaacc cagcgctggg tcggtgctga ggcattgttg cgttggaagc atccggtgtt gggtgaattt tccaatgaaa ccttgattca tatggcagag caaaatggtc ttatctttga agtggggcat tttgttttgc accaagcttt aaaagccgcc agtgattggt tagcggtgtg cccaaccttt tgtatcgcga tcaatgtctc ttccgtacag ctcaaaaaca gtggctttgt cgagcagatt cgagatctgc tggcgctgta ttgcttccct gcgcatcagt tggaactgga aatcaccgaa agtggcctga tcgtcgatga gccgaccgcg agtgatattc tcaaccgact acacacatta ggcgtgacat tatcactcga tgattttggt acgggttacg cttcgtttca gtatctaaaa aaattcccat ttgatggcat caagattgat aaaagtttta tggagcagat cgaacacagc gaaagcgatc aagaaatcgt gcgttctatg ctgcatgtag cgaaaaaact gaacttaaac gtggtggtgg aaggtattga gtcgacgcag caagagcagt tcattctgga acagggttgc gatgtcggcc aaggcttttt atatggcaaa cctatgccca gtgaagtgtt taccctcaag ctcgaaagcc acgctctggc gtaa SEO ID NO: 42 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31635.1) SEO ID NO: 43 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (GI:695934490 REGION 491690..492670) ttagaaaagt tcaacgtcat cagaaaatgg ccgttgcgcg ctggcaattt taccgttctc acacagctgt tcatagcagt gcacctgatt ccgaccatgc tctttggcgt aatacaacgc tttatcggca tggtcgagaa tggtaggtaa atagtcaccc ggcctgagtg agcaaaaacc agcgctgaag ctcagttcac cgattctcgg gaagttatgg cgtcggatct gttgacggaa gccatccaac tgttgcttga tttgtggctc attaccgctt gaaaaaataa tcacgaactc ttcaccacca aagcgaaata gttgagaaga cggtccgaaa tagtgctgca tctgctgagc gaacataagc agaatttcat caccaatcat gtgtccgaag tgatcattga tcgctttaaa atggtcaata tccaacatcg cgatccagag tttgtgattc tcttctgtcg agggattgat ggcaaaggtg tggcgcaatc ggtcttctaa cgttcgacga ttgagtaatc cggtcagctt atcgcgttca ctctcatgca aaatcaccgt gtaattacgg taaattttcg caaatccgtt gatcaacatg cgataaggtt caggatcttt attgaggatt aagcacagct ctgcggaaaa gtgttcttct atcggaatcg ggcaaaagca ttgatattgg ccattcgctt gttgggaaaa cgccatttcc gattgagagt gctggtaacc attgtcggca catacttggt cgtattgcca ctggtactcc tttttacctg cagcattttt ggtaataatt aaacgtgcca ccataagggt tgaacgtcca agatggtgaa ataaggtcgc cgtggagagc ggtaacaatt cagacaaggt cgccaaaata ctgtaactga gtgccagcga atttttctgc tcagtaattt caataaccga ctcaagcact ttgtcattca t SEQ ID NO: 44 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31689.1) SEO ID NO: 45 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (GI:695934573 REGION 592066..592992) atgatagaac ttaatagaat tgaagagctt tttgataacc aacagttctc cttgcacgaa ctcgtgttga acgaactggg agtctatgtc ttcgtcaaaa atcgccgcgg cgagtatctc tatgctaacc ctctgactct aaagttgttt gaagcggatg cacaatcgtt gtttggcaag accgatcacg atttttttca tgatgatcaa ctcagtgata tcttggcggc cgatcaacag gtgtttgaaa ctcgtctctc ggttatccat gaagaacgag ccatcgccaa atccaatggt ttggttcgga tttatcgcgc agtcaaacac cctatcttgc accgagtgac aggcgaagtg attgggctga ttggagtttc aaccgatatc accgatatcg tggaactgcg tgagcagcta tatcagctcg ccaataccga ttctttaact cagctgtgta atcggcgtaa attgtgggcc gattttcgcg ccgccttcgc tcgcgcaaaa cgtttaagac agccgttaag ttgcatctct atcgatattg ataatttcaa actgatcaat gaccaatttg gtcacgataa aggtgatgaa gtcctgtgtt ttctcgccaa actatttcag agcgtcatct ctgaccatca tttttgtggt cgtgtgggag gtgaagagtt catcatcgtt ttggaaaata cgcacgtaga gacggctttt catttggctg aacagatccg ccaacgtttt gcagagcatc cgttctttga acaaaacgag cacatctacc tctgtgcggg ggtttccagc ttgcatcatg gtgatcatga cattgccgat atttatcgac gctccgatca agcactgtat aaagccaagc gtaatggtcg taaccgttgc tgtatctatc gccaatccac agaataa SEQ ID NO: 46 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31772.1) SEQ ID NO: 47 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence CGI:695934589 REGION 606596..607612) atgacaactg aagatttcaa aaaatccacg gctaacttaa aaaaagtcgt acctttaatg atgaaacatc atgtcgcggc cacccccgtg aactatgcct tgtggtatac ctacgtcgac caagccattc cgcaactgaa tgcggaaatg gactctgtat tgaaaaattt tgggctttgc ccacccgctt ctggtgaaca tctttaccaa caatacattg cgaccaaagc agaaaccaat attaatcagt tacgtgcgaa tgttgaggta cttcttggtg aaattagcag ttcaatgagt gatacgctca gtgacaccag ttcctttgct aatgtgattg ataaaagctt taaggattta gagcgcgtcg agcaagacaa tctctcgatt gaagaagtaa tgacggtgat ccgccgcttg gtgagtgact ctaaagatat tcgacactca accaatttcc taaataatca actgaacgcg gcaacactag aaatctctcg tcttaaagag cagctggcga aagttcagaa agatgctctg tttgacagtt tatctggact ctataaccgc cgagcttttg atggcgatat gttcacgctg atccatgcag gtcaacaagt cagcctgatc atgctcgaca tcgaccactt caaagccctt aatgataact atggccacct gtttggtgac caaattatcc gtgcgatcgc caaacgtctt caaagcctat gccgtgacgg cgtgacagct tatcgttatg gcggtgaaga gtttgcactg attgctccgc acaaatcgct gcgtattgca cgccagtttg ctgaatcggt gcgacgttca atagaaaagc tcaccgtaaa agatcggcgt agcggtcaat cggtcggtag cattaccgct tcgtttggtg tagtagaaaa gattgaaggt gactctttgg agtctcttat cggtcgagcg gatggattgc tgtatgaagc gaaaaatctg ggccgcaatc gagtcatgcc gctctaa SE0 ID NO: 48 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31788.1) SEQ ID NO: 49 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (G1:695934592 REGION 610255..611628) tcaaaagcga tagagtgggt tttgcctacg cttagcggta tacatacgtt catcggccag tttgaacatt tcatcaggtg tggcaaacga ctggtcatac aaagcatatc cgatacttac acgaacatgg ataagcttgt cgtcataaac gatgggcgtt tcagaaatcc tttttaaaat attgtcactg actttaagca cgtcttgttc acgatgaatt cgtggaatta acacgagaaa ctcatccccc ccaatccgcg ccaccagatc ggaaacccgc aggctcgatt taattctttc cgcacaagcc accagcactt tatcgcctgc gctatgtcca tgggaatcgt tgatagattt aaaacggtca atatcaatgt tcaacaaagc aaagttacct tcgctatgag agcgcttagc attttcaaag tagtgttcaa tggtatagat aaaatagcgc cgattcggca agtgggttaa agggtcatgt agcgcacgct cctccgcgac ttgataaagg cgcatgataa cgccaaagcc tgccatcaat accaataaca ccgagtatcc caacaagcgc actgcatttc gggtatacca agataactgc tgtagtaaat cttgcttttc agcgaccgca attcgccaac ttccgtaagg gaaatagaca ttctcttgtg caaaagcgtg ctcaaatact cgaggctctc caaaaaacac gtccccctca ctgccacggc tgtctaaacc acgaatcgca acctgaaaat gctccccaaa gctgtaaata ctggttgctg aaagcaatga atcccaatcc atcaccacac tcagtacccc ccaataacgc gtatccttcg qtgggtcgta gaatatcggt tctcgaatca ccagcgcgcg cccaccttga acgagatcga caggtccaga gacgaacgtc tgtttgattt cacgtgcttt ttttattgac tgccactgct gaggaacggt gcggtaatcc aaaccgagta gtgcattggt ttgaggaagc ggatagctga aagcgaccac atcattaggg gcgataccta atgagcgtaa gtgatcgcta ttcctgatca ccgccgctga aagcggctcc cattgataga tattgaggtc gggatctagg gttaacaggg ttgttaaacc ttttacggta tagatatcac ccaaaatctc agcttctaat tgaaaacgta cgatggaaag atcttcttta gcttgttgac gtaaaccctc ttgtagatca cgtgtatggc taatatgaag ggattcaata accgcaatgc ccaaaaagag taaggcgaga aaataaattg agacatactt atatttgtgc gaggttaacc ccat SEO ID NO: 50 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31791.1) SEQ ID NO: 51 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (GI:695934597 REGION 616194 .617369) atggatagct ttgctggcaa ccaattaaaa gagatgacag agatgcgttt tgctcgtaag cagcatattg tcctgatcag ctctggtgtt gctaccgcta tttttcttgg gtttgccctt tactactatt ttaaccatca acccctgtca tccggtttat tgttattaag cggtattgtc accttattga atatgatttc gctgaatcgt caccgcgaat tacacactca agccgattta attctgtcat taattctgct cacttatgcg ctggccttag tcagcaatgc tcagcatgaa ttatcgcatc tcttatggtt atatccgctc atcaccactt tagtcatgat taaccctttt cggttaggct tggtttacag tgcagcgata tgcttagcga tgaccgcctc tatccttttt aatccggcac aaactggctc gtaccctatt gcacagacct attttttagt aagtctattt acgctgacga ttatctgtaa taccgcttct ttctttttct caaaagcgat caattatatt cataccctat accaagaagg tattgaagag ttggcttatc ttgatccgtt aacgggctta gccaatcgtt ggagctttga aacttgggcc acagaaaagc tcaaagaaca acagagttcg aataccatta ccgcgcttgt ttttctggat attgataatt tcaaacgcat taatgacagt tacggccatg atgttggcga tcaggtgtta aaacattttg cacaccgtct acgcaataat attcgtaata aagatcgagc caccaatcaa catgattatt ccattgctcg atttgctggt gatgagtttg tgctcttgtt atatggtgtg cgaaatttgc gtgatctcga taatattctc aaccgtatct gtaatctctt cgtcgaccgc tatcctgaga cggatatgct caacaacctc acggtgagta taggggcagc tatttatccc aaagatgcga tcactctgcc ggaactaacc cgctgcgcag ataaagccat gtatgccgct aaacacggtg gaaaaaatca gtaccgctat taccatgatg ccgctttccc tccggctgta gaaaccgtat taggcagtca gcccgttgag gctcctaacg taactccact gaaaaaagcg cactaa SEC) ID NO: 52 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31796. 1) 5E0 ID NO: 53 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (G1:695934700 REGION 737143..739053) atgacgctat acaaacaact agtcgcaggg atgattgcgg tgtttattct gttgttgatt tcggttttta ctatcgaatt caacaccact cgcaacagtc ttgaacaaca acaacgctct gaagtcaaca acaccataaa tacggtgggt ttggctttag cgccttatct ggagaagaaa gacaccattg cggtagagtc agtcatcaat gcgctgtttg atggcagtag ttactcgatc gtacgtctga tttttctcga tgacggtacg gaaatcctgc gctcataccc tatccaaccc aataatgtgc cggcttggtt tactcagtta aatctgtttg agcccatcca tgatcggcgt gttgtaacca gtggttggat gcaattggcg gaagtggaaa tcgtcagcca tcctggtgcg gcttacgctc aactctggaa agcattaatt cgtttaagta tcgcgttttt ggcgatctta gtgattggta tgtttgccgt cgccttcatt ttgaagcgct ctctaagacc actacaactc atcgtcaaca aaatggagca ggttgctaac aaccaatttg gtgagcctct accgcgcccc aacactcgag atctgattta tgtagtagat ggcatcaata agatgtctga acaggtcgag aaagcgttta aagcccaagc caaagaggcg cagcaactgc gtgaacgtgc ttatcttgac ccagtttctc atcttggcaa ccgagcatac tacatgagcc aattgagtgg ctggctctct gaaagcggca tcggtggtgt agccattcta caagctgaat tcatcaaaga gctttatgaa gagaagggct atgaagccgg tgatggcatg gtgcgcgaac tggcggatcg ccttaaaaac tccatcacca tcaaggacat ctctatcgct cgtatctcca cttacgagtt cggtatcatc atgcctaaca tggatgaaac tgagctcaaa atcgtggcag agagcatcat cacttgtgtg gacgacatta accctgatcc tactggtatg gcgaaagcca atttatcgct tggcgtggta agcaataagc gtcaatccag caccacaacg ctcttgtccc tgctggataa tgcgttagct aaagcgaaat ccaatcctga gctgaactac ggctttatta gcagtgatac tgataaaatc atcttgggca aacagcagtg gaaaactctg gtcgaagagg caatccataa cgactggttt actttccgct accaagccgc caacagcagt tggggaaaaa cattccatcg cgaggtcttt tctgcgtttg agaaagacgg cgtgcgttac acggcaaacc aattcttgtt tgcccttgaa cagctcaatg ctagccatat cttcgatcag tacgtgattg aacgtgtgat tcaacagctt gaaaaaggcg aactgaccga tccactcgcg atcaacatcg cacaaggcag tatctctcaa ccgagcttta tccgttggat cagccaaacc ttaagcaagc atctttctgt ggccaactta ctgcattttg agatcccaga aggctgtttc gtcaatgaac cgcattacac tgcgctattt tgtaacgcag tacgcaatgc aggggcggac tttggggtag acaactacgg acgtaacttc caatctctcg actacatcaa cgagttccgt cctaaatacg tcaaactgga ttatctattt actcaccatt tggatgatga acgccagaaa tttaccctga cctcaatctc gcgcaccgcg cataacttag ggatcaccac catcgcatca cgggttgaaa cacagactca gctcgatttt ctttcagaac atttcatcga agtcttccaa ggcttcattg ttgataagta a SE0 ID NO: 54 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31899.1) 301 sITIKDISIA RISTYEFGII MPNMDETELK IVAESIITCV DDINPDPTGM AKANLSLGVV

SE() ID NO: 55 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (GI:695934774 REGION 830662..832242) ctactcaaca cacacttggt tacggccatt ggctttggcg cgatacaaag ctttgtcagc gcggtagaac gtacgttggg tattttcccc ctcgcgatgc aaggtgatac cgatactgac cgtcagtccc cgttcgccaa gtacgtcttg ccatgggaaa tcaaaaatac gttggcgata ggtttcggca tgcatttgtg ccatatcact ggtgacgttt tccaaaatca ccagaaattc ctcgccaccg aaacgtacgc aggaggcacc acggaattta aagtaactcg ccagttcact ggatacattg acaatcgctt tatcccctac caaatgactc aattcatcat tgatcgattt aaagtggtca atatcaacga ctaagaaagc aaacggggtt tcgtgcagca gcagatcttt cagcttcacg tccaaccaac ggcggttatg cagttttgtc agtggatcgg tgaacacatc ttgctgtagt tgcaacaccg tattcttctg gctttcggtg gtttctttta gctcacgatt ttctaattcc gacaaaatca gtttaagttg tagctcaaag cgcgataggc ggcgtagctg aattgggcct aattcactga tggggatccg cttcatcaaa tcgctttcga tgcgaaatgc tttcttttcg taaaccagtg cggttttgta cattccttcg agtttacaca cttcgctgaa cgcttcatag aggcgttttt caaggaaagg ggaatgaatg ttttgtaagc gcttttcagt gctacccagc agcatggtgg caaaatgcgc cttacctgct ttagagaggc aatgcgctaa ctcgatgcgt agcatgcttg atagccaatc cgatggcgtc agcgatgacg aatactgtgc attggcgagt gtcatcatcg ccetttgcac tttgccttgt tgcagataaa gcttggcttg atagagcatg atctgcccag tcagcagttt atcgctgacc agaatgctca actcatcaca ctcttttatc agatcattgg ccgctgcata acgaccaagg ctgatgtagc aagccagcat atacagcttg taacgcaggc gcagtgagcg gctagaaatc gcatgatcta tgctgtcaat tttttggtag tagcgtaacg cacggctgtg atcgccataa gcatcacata aattgcccat tccgagcact gcaagtacgt agtcatcaat catgccatgc tcaacggcga tgttggatat cgcaacgtat tcagacagtg ccgcgacata ttcaccatgg tcgagtaaac gctcactcaa actgtgtttg accgagagca ttaattccag atccgtcggt aactctaata gggaaagagc ggcgcgcagc tcttcaatac tggtttgcca ctgtttcatt tcgcggcggt attcggcgct gatgatgtag ctttgtgcac gctcttgggc ggtggttgcc acgtgctgtc tgacatggtt ccagaaaatg atcgcctctt caccagcgat agcggccgca tccagtcccg cttctttgat cttattgagc agggtttcca t SEO ID NO: 56 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31973.1) 301 ALVYEKKAFR IESDLMKRIP IsELGPIQLR RLSRFELQLK LILSELENRE LKETTESQKN

SEC) ID NO: 57 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (GI:695934794 REGION 857071..858171) tcacgatgag gggctttttt gtaggaattt catttcatac atgtttttat ctgccagatg gatcaactgg ctcaaattgg tgctgtcgag tggataagta ctgaccccga cgctggtgtt gagcttggct cgtaaatcgc cacttaattc aaattcatgg tcgaaacact gtttgatcat gcgctgcatc atcatctgct cggtcgaatt gatgctgctt aggatgatgg caaattcatc tccccccatc cgaaacacac gataatcgaa tgaaggaatc gagttgttta agcgataagc aacctgtttg agtaccgcat cgcccatttg atggccgtag gtatcattaa tttgtttaaa accattcaga tcgagcaaaa agagagagaa tccaccgctg cggcggtggc gttctaattc ggcgaacatg gctgtgcggt tttccagccc tgttaatggg tccgttaagg ccaagactct atggtgcgtg gcctctttat gcaaaataaa actcaccagt cccacacagc taaacgtcaa caaaattaac gcaaactgga tgcgactgag gtaattcagt ttctcttttt gctctacata caaaggactt tgcattccaa atgtgcggtt tatgaactga ataaaaatct ccagctcttg ttgggcggca acaataaaag tttgtaagct ttctggattt ttggccgcaa gcagtagcgg ttcaagttgt ttaaagcgcg caaacgcggc ttggaagaat tcgcgagtgc tgggcatgcc tataatgccg tcggcttctg ggctattgag gatcagatca aaacggctcc aagtcagctc atatttcacc atcacatcgc gctggttgct ctccgactcc aataggtagg gggagagtgc cagcatctca gtaaactctt tattgagctg gaataagaac cagatcgctt ggttagtatg cgaagagtaa gacttagata aatcgcgagt actgttgatc aaatacaaat tggccaaaat cagaatcgcc gacatgaaga tcagcagtgt tttggcatgt aagatcagcg ggtggagcgt tttctgagtt tgtgtattca t SEQ ID NO: 58 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31993.1) SEQ ID NO: 59 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence IGI:695934800 REGION 864637..866460) ttaggctaca ttcgtttctt ttctccagcg ttcaatcatc acactcggta aatcaggtcg actgaagtaa tacccttgaa tttgctcaca gcccatttga tagagtttat ccagtgcttg ttggttctct accccctcag cgacgagatc gagtttaagc tggttagcaa gctgaataat caaccacacg atactctcag aggtttggtt ggtaagtagg ttacgcacaa atgcagcatc aatcttgatg caatcaatcg gataactgtg aatgtagtta aggctcgaat aacctgtccc aaaatcatcc aaggcaattt taaaacccaa ttcacgcaat atggtgagaa tactgcatac ttctgcggcc ttagagagta aaaccgtttc tgtcagctca atagtgaact cgtcggcttg aaaaccatag gctttaatgg tttttaatag atgctcaagg taacgattgg aatgcgtcag ctcatcggcg gagcagttga tgcttaagcg aattttttgg tcaatacctt gttctaattc ttgtttcgcg atgcaggcca attcgagaat acgttcgcca aattcgacaa tcaggccaga ttgctctgct gcttcaatga attccaatgg cgttaccaca ccgagcgtac tgctattcca acgcgttaag atctcaaaat agtcccaatt tctttgatgt tttttcacga tcggttgcac gaccacatac agctcagttt gatggatagg cttactcaat tcactacgca gagcttcgat gatttgtgta cgccgatagt attgattgct gagtaagttg tcgtagaaac gaatgcgtgt gttatggttc cgtttacact cttttaaagc gagacttgca ttgaacagta attgatcggc attgagcttt tcaccactgt atttggtaat accaatactg acactgattt tgagtcgacg atcttgatcg atataatctt gcgccagctt gttgagtatg gtttggcaga tcttcatcgg ctcacgatct gtggttaaaa aagcaaattc atcagcggcg attcgaaagg cgtatccttc ttcggggacg gcttgtttta tcgcatccgc gacaaatttc agcacaagat ctcccaaata gtgcccatgc agatcgttta tcgaacgaaa ttcatcaata tcaagaaagg ccagagtgaa atgatgtcta tcttcttgaa cgagagccgt cagtttctcg gctaaatcat tacgattcat taaacccgtt aagttgtcgt gagatatttc atgacgtaat tgattgatta ggctctgaga gcgtacctcc atctgtttac attccagatc atgagcgatc atctgagcca aaatctggtg aactaacacg agattagcaa agtcgtctaa ctgacgcgta aaagtcgaga tcaaaacgcc gtagttttcg ccatttgaaa aataaatcgg gatacccaga tacgcctcaa tatggttctc aactaaataa gcatcgttag gaaaaagttc cgcgactttg cttgcaaata ggcaataagg ttgtctttgt aatccgactt gctcacaagg tgtgccttgt agttcgtaat acagctctaa actgctgggt tcgacactgg cacaacttaa gttatgagct ttgtagcgca ttttatctag ctcaatgacc attgagctgt ggctattgaa ggtgcggtgg agaaactgag tgatttgtga gagcaactcc aaccccccca gctgactgaa gtgatgtatg gaatctaggc tcagtttttc tgttatcagt tgagtcttgg tcat SEQ ID NO: 60 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT31999.1) SEQ ID NO: 61 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (GI:695934874 REGION 956091..958088) gtggcaggtc acaccttact ctcttccaac acgtttacgc cgctagaagc gtatcctgaa gccttttggg catgggctgc gcagtttgat acttccgatg gtttgatccc ttttgccatc aatacctgtc gctggaacta tttgccagtg atgggcggtg agtcgtttat ttttatgctg gataatcatc ctcagcatcg gacttatctg atcattcaag cggcatgcgt cgataaagta cacctgagca ctcaatccgg tgagttggat tttttacagt taattgcagc gaaatggcaa tgcttacgag cggaaattga agcatcgaaa gagtttaaaa atcgtgattt acgtgaggcg cagtacctta gtgaaattcg tcagcgagag cagtttattg acaacatgaa gctggtgcat caagtcgcgc tcgagttgtc caaccccgcc aatcttgatg agctacaccg cgcatcggtc gaggctatgc gacatcgtct cgggtttgat cgatccgcgc tcttgttgct tgatatgaaa aagcgttgct tcagcggtac ttatggtacc gatgagcacg gtaatacgat tgatgaacag cacacccagt atgatctgca ccaattagag cctcaatatc tcgaagcttt atccaatgaa gagtgcactt tgatggtggt ggaagatgtg cctttgtaca ccgtcggaca ggtagtggga caaggctgga atgccatgct gattttgcgt gatggtaatg acaccatagg ctggattgcc atcgacaact atatcaatcg gcagccgatt accgagtatc aaaagcagat gcttgagtcg tttggctcat tgctcgcgca aatttatatt cgtaaaaagc aggaacaaaa cgtacgtatg ctgcatgcca gcatggtcga actgtctcgc tgtatgacag tcagtgaagt gtgtaaatcg gcagtcacct ttgcgatcaa ccgaatgggg attgatcgca tggcggtgtt tttgacggat gaagcttgct cttatattca ggggacgtgg gggacggata ttcaaggcaa tattgtcgat gaatcctatt tccgtggttc aacgcatgaa aatgacattg tcgaccttgc caaagtgtac ccaaacgaag tggtgtttaa agagagtgtt cccatctatc acgactgtaa aattgtcggt tatggttgga cggcgatgac catgctcacc gacaaaggca ccccgattgc ctttattgcg gcggataatt tgatccgacg ttcccccttg acttcacaac tgcgtgaagt gattcgtatg tttgcttcaa acctcaccga agtcttgatg cgagccaaag cccaagaagc gatctcggta ctcaatgaaa cgctggagct tgaggtgcgt aatcgcactc gtgatttgca aaaggccaac gaaaaactcg atttaatggc gaaattagat ccgctgactc gtttagggaa tcgccgtatg cttgagcacc aactggagca aacttgcgaa cagaccatca aagaggtggt caattatggc gtgatcttgc ttgatattga ccatttcggg cttttcaaca actgctatgg tcatcttgaa ggcgatattg ctctgatgcg gattggtaat atcctcagtc gacatgcgca atctgagcat gaactgttct gtcgtattgg tggggaagag tttctgcttt tagtcgccaa tcgaagcgcc gaggagattc acttactggc tgaaaatatt cgtaaaagta ttgaagcaga atgcattgaa cactgcgaaa atcccagtgg tgagctactg accgtatcga ttggttatgc tgcttctcgt tataaaccgc gagagattca atttgatcag ctctatgcag aagcggataa agccttgtac agagcgaaaa gccaaggacg gaatcaggtt attggcgtta ttgttgaaaa tatcgactgc atacaggcag aaatgtag SEQ ID NO: 62 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT32073.1) SEQ ID NO: 63 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (G1:695934896 REGION 980640..981086) atgctagcgt tacctgcgga gtttgagcaa ttccattgga tggtcgatat ggttcagaat gtcgatatgg gattgattgt gattaaccga gactacaacg tgcaagtgtg gaatgggttt atgacccatc atagcggtaa gcaagctcat gatgttattg gtaaatctct gttcgagatt tttccagaga tccctgtgga gtggtttaag ttaaaaacca aaccggtgta cgatctgggt tgccgtagtt ttattacttg gcagcagcgc ccttatttgt tccattgccg taatgtgcgc ccagtgactc agcaagccaa atttatgtat caaaacgtca cgcttaaccc aatgcgtaca ccgacaggcg cgataaattc actcttctta tccattcaag atgcaacaag tgaagccctt gtttctcaac aagcttcttc tcaataa SEQ ID NO: 64 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT32095.11 SEQ ID NO: 65 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (GI:695934918 REGION 1008191..1009270) tcagcgatga ccatgagttg aacccaatag cgcatgacaa tggtcaccat tgagttcaat gacatgctct tcatcgaagc tgacgcggtt tttccccatt tttttcgaat gatagagagc ttggtctgcg cgtttgaacc actgctccgg atcatcggtg cgaagtgctt cggctaaacc gacactgacg gtgactttgg catggtatgg gtagtgcgtt tgttgaatcc gacaaccaat atgactcatc acgagtgtag cgtcggttaa cgacgtattt tcaaacagca gtaaaaattc atcgccccct aatcgaaaca acagatctaa ctcacggcag tgagtattca ttatttcaac aacttgggta atgactttat ctcctgtgtc gtgtccataa aggtcattaa cagatttgaa gtgatcgata tcgatcacgg cgatcaccgc cgattcattg gcgagctggc ggtggcgaag acattttttc aaaaaaccat ccagttgatg acgattcaat gtgcccgtta atgcatgacg agtggaaaga taaaaaagct cagtgtgcag cttacggata gcatctacca ccacatacat gatggcggca caagcgctga tcgcaaggct aaagcgcaag gtgacttcgg cggtttgatg gggaattaaa acccatatgc tggctggaat gataatggtg atggtcaata agttatcttt ctgggggagt agaaaagcaa tcgcaatgag cacgggaaat agccagtagc tggcgagggt gccgaaaatg tgaatagcca tcaccacgat gactaccacc aatgccagtg gaagcctaaa accccatggt gttttctttt gataatagat agccgtaatt tcaatgagga gcgtgcattg gaatacgatg atcaacccgc caagaagaac gtagtcaatc agcaagtttt taacggcgag tggaaagaaa accaaactag aaataaaacc aataaaaagc gacacccgac gttgatagta agtgttcagt aactctgaac cggtaaaagc aggagagtga gtcgattttg tcatcgtcat , WO 2017/049127 SEQ ID NO: 66 Vibrio cholerae strain 2012EL-2176 chromosome 2 amino acid Sequence (AIT32117.1) SEQ ID NO: 67 Vibrio cholerae 2012EL-2176 chromosome 2 DNA Sequence (GI:695934235) atggatcatc gcttttcgac caaactgttt ctgcttctca tgattgcttg gccgctttta ttcggatcaa tgagtgaggc tgtagagcgc caaaccttga ctattgccaa ctcaaaagca tggaaaccct attcttattt ggatgaacag ggacagcctt ctggcatatt gattgatttt tggttggctt ttggtgaagc gaatcatgtc gatattgaat tccaactgat ggattggaat gattccctag aagcggtgaa gcttggcaaa tccgatgttc aagctggttt gatccgttct gcttcaagat tagcgtatct cgattttgca gaacctttac tgacaatcga tacacaactc tacgtacacc gcacgttatt gggcgataaa ttggatacgc tgctatcggg ggccattaac gtctcattag gtgtagtaaa agggggattt gaacaagagt tcatgcaacg agaatatcct caacttaagt tgattgagta cgccaacaat gaattgatga tgtctgcagc aaagcgacga gaattagatg gttttgtggc cgatactcag gtcgccaatt tctatatagt ggtttccaat ggcgcgaaag attttacgcc agtgaagttt ctttattcag aggaattacg tccagcggtc gccaaaggca atagggattt attagagcaa gtagagcagg ggtttgcaca attaagtagc aatgagaaaa accgtatttt aagtcgatgg gttcatattg aaacgattta tccacgttac ttaatgccga ttctcgcttc aggtctctta ctcagtatcg ttatttatac tcttcagcta cggcgtaccg ttcgattgcg aacacagcaa cttgaagaag ccaatcaaaa actctcctat ttagcgaaaa cggatagctt gacggacatt gctaatcgcc gttcgttttt tgaacatctt gaagcggaac aaacacgatc aggcagctta acgttgatgg tttttgatat tgatgacttc aaaaccatta acgatcgctt tgggcatggc gcaggagata atgccatctg tttcgtggtt gggtgtgtgc gacaagcttt agcatcggat acctactttg caaggattgg tggtgaagag tttgctattg tagcgcgtgg taaaaatgca gaagagtcgc agcagttagc tgagcgaatt tgccaacgag ttgcagaaaa aaagtgggta gtgaatgccc aacactctct gtcactcacc atcagcctag gctgtgcatt ttacctacac ccagctcggc cattcagttt gcacgatgcc gatagcttaa tgtacgaagg aaagcggaat ggaaagaacc aggttgtctt tcgtacctgg tcataa SEQ ID NO: 68 Vibrio cholerae VCA0848 01 biovar El Tor. str. N16961 chromosome II DNA Sequence (gi115600771:c790898-789918; NC 002506.1) ATGAATGACAAAGTGCTTGAGTCGGTTATTGAAATTACTGAGCAGAAAAATTCGCTGGCACTCAGTTACA
GTATTTTGGCGACCTTGTCTGAATTGTTACCGCTCTCCACGGCGACCTTATTTCACCATCTTGGACGTTC
AACCCTTATGGTGGCACGTTTAATTATTACCAAAAATGCTGCAGGTAAAAAGGAGTACCAGTGGCAATAC
GACCAAGTAT GT GCCGACAATGGT TACCAGCACT CT CAATCGGAAAT GGC GT TTT
CCCAACAAGCGAATG
GC CAATAT CAAT GCT T TT GC CC GAT T C C GATAGAAGAACACT TT TCC GCAGAGC T GT GCT
TAAT CCT CAA
TAAAGATCCTGAACCTTATCGCATGTTGATCAACGGATTTGCGAAAATTTACCGTAATTACACGGTGATT
TTGCATGAGAGTGAACGCGATAAGCTGACCGGATTACTCAATCGTCGAACGTTAGAAGACCGATTGCGCC
ACACCTTTGCCATCAATCCCTCGACAGAAGAGAATCACAAACTCTGGATCGCGATGTTGGATATTGACCA
TTTTAAAGCGATCAATGATCACTTCGGACACATGATTGGTGATGAAATTCTGCTTATGTTCGCTCAGCAG
ATGCAGCACTATTTCGGACCGTCTTCTCAACTATTTCGCTTTGGTGGTGAAGAGTTCGTGATTATTTTTT
CAAGCGGTAATGAGCCACAAATCAAGCAACAGTTGGATGGCTTCCGTCAACAGATCCGACGCCATAACTT
CCCGAGAATCGGTGAACTGAGCTTCAGCGCTGGTTTTTGCTCACTCAGGCCGGGTGACTATTTACCTACC
ATTCTCGACCATGCCGATAAAGCGTTGTATTACGCCAAAGAGCATGGTCGGAATCAGGTGCACTGCTATG
AACAGCTGTGTGAGAACGGTAAAATTGCCAGCGCGCAACGGCCATTTTCTGATGACGTTGAACTTTTCTA
A
- 59.

SEQ ID NO: 69 Vibrio cholerae strain 01 biovar El Tor str. N16961 amino acid Sequence (NP 233234.1) SE0 ID NO: 70 Vibrio cholerae strain 01 biovar El Tor str. N16961 Vc DncV
DNA
Sequence NC 002505.1, gi115640032:180419-181729) GTGAGAATGACTTGGAACTTTCACCAGTACTACACAAACCGAAATGATGGCTTGATGGGCAAGCTAGTTC
TTACAGACGAGGAGAAGAACAATCTAAAGGCATTGCGTAAGATCATCCGCTTAAGAACACGAGATGTATT
TGAAGAAGCTAAGGGTATTGCCAAGGCTGTGAAAAAAAGTGCTCTTACGTTTGAAATTATTCAGGAAAAG
GTGTCAACGACCCAAATTAAGCACCTTTCTGACAGCGAACAACGAGAAGTGGCTAAGCTTATTTACGAGA
TGGATGATGATGCTCGTGATGAGTTTTTGGGATTGACACCTCGCTTTTGGACTCAGGGAAGCTTTCAGTA
TGACACGCTGAATCGCCCGTTTCAGCCTGGTCAAGAAATGGATATTGATGATGGAACCTATATGCCAATG
CCTATTTTTGAGTCAGAGCCTAAGATTGGTCATTCTTTACTAATTCTTCTTGTTGACGCGTCACTTAAGT
CACTTGTAGCTGAAAATCATGGCTGGAAATTTGAAGCTAAGCAGACTTGTGGGAGGATTAAGATTGAGGC
AGAGAAAACACATATTGATGTACCAATGTATGCAATCCCTAAAGATGAGTTCCAGAAAAAGCAAATAGCT
TTAGAAGCAAATAGATCATTTGTTAAAGGTGCCATTTTTGAATCATATGTTGCAGATTCAATTACTGACG
ATAGTGAAACTTATGAATTAGATTCAGAAAACGTAAACCTTGCTCTTCGTGAAGGTGATCGGAAGTGGAT
CAATAGCGACCCCAAAATAGTTGAAGATTGGTTCAACGATAGTTGTATACGTATTGGTAAACATCTTCGT
AAGGTTTGTCGCTTT.ATGAAAGCGTGGAGAGATGCGCAGTGGGATGTTGGAGGTCCGTCATCGATTAGTC
TTATGGCTGCAACGGTAAATATTCTTGATAGCGTTGCTCATGATGCTAGTGATCTCGGAGAAACAATGAA
GATAATTGCTAAGCATTTACCTAGTGAGTTTGCTAGGGGAGTAGAGAGCCCTGACAGTACCGATGAAAAG
CCACTCTTCCCACCCTCTTATAAGCATGGCCCTCGGGAGATGGACATTATGAGCAAACTAGAGCGTTTGC
CAGAGATTCTGTCATCTGCTGAGTCAGCTGACTCTAAGTCAGAGGCCTTGAAAAAGATTAATATGGCGTT
TGGGAATCGTGTTACTAATAGCGAGCTTATTGTTTTGGCAAAGGCTTTACCGGCTTTCGCTCAAGAACCT
AGTTCAGCCTCGAAACCTGAAAAAATCAGCAGCACAATGGTAAGTGGCTG.A
SE0 ID NO: 71 Homo sapiens Mab-21 domain containing 1 (MB21D1), Human cGAS, transcript variant Xl, rnR_NA (XM 017010232.1) 1 gcgacttccc agcctggggt tccccttcgg gtcgcagact cttgtgtgcc cgccagtagt 61 gcttggtttc caacagctgc tgctggctct tcctcttgcg gccttttcct gaaacggatt 121 cttctttcgg ggaacagaaa gcgccagcca tgcagccttg gcacggaaag gccatgcaga 181 gagcttccga ggccggagcc actgccccca aggcttccgc acggaatgcc aggggcgccc 241 cgatggatcc caccgagtct ccggctgccc ccgaggccgc cctgcctaag gcgggaaagt 301 tcggccccgc caggaagtcg ggatcccggc agaaaaagag cgccccggac acccaggaga 361 ggccgcccgt ccgcgcaact ggggcccgcg ccaaaaaggc ccctcagcgc gcccaggaca 421 cgcagccgtc tgacgccacc agcgcccctg gggcagaggg gctggagcct cctgcggctc 481 gggagccggc tctttccagg gctggttctt gccgccagag gggcgcgcgc tgctccacga 541 agccaagacc tccgcccggg ccctgggacg tgcccagccc cggcctgccg gtctcggccc 601 ccattctcgt acggagggat gcggcgcctg gggcctcgaa gctccgggcg gttttggaga 661 agttgaagct cagccgcgat gatatctcca cggcggcggg gatggtgaaa ggggttgtgg 721 accacctgct gctcagactg aagtgcgact ccgcgttcag aggcgtcggg ctgctgaaca 781 ccgggagcta ctatgagcac gtgaagattt ctgcacctaa tgaatttgat gtcatgttta 841 aactggaagt ccccagaatt caactagaag aatattccaa cactcgtgca tattactttg 901 tgaaatttaa aagaaatccg aaagaaaatc ctctgagtca gtttttagaa ggtgaaatat 961 tatcagcttc taagatgctg tcaaagttta ggaaaatcat taaggaagaa attaacgaca 1021 ttaaagatac agatgtcatc atgaagagga aaagaggagg gagccctgct gtaacacttc 1081 ttattagtga aaaaatatct gtggatataa ccctggcttt ggaatcaaaa agtagctggc 1141 ctgctagcac ccaagaaggc ctgcgcattc aaaactggct ttcagcaaaa gttaggaagc 1201 aactacgact aaagccattt taccttgtac ccaagcatgc aaaggaagga aatggtttcc 1261 aagaagaaac atggcggcta tccttctctc acatcgaaaa ggaaattttg aacaatcatg 1321 gaaaatctaa aacgtgctgt gaaaacaaag aagagaaatg ttgcaggaaa gattgtttaa 1381 aactaatgaa atacctttta gaacagctga aagaaaggtt taaagacaaa aaacatctgg 1441 ataaattctc ttcttatcat gtgaaaactg ccttctttca catggagtct cgctctgtcg 1501 cccaggctgg agtccagtgg catgatcttg gctcactgca agctctgctt cctgggttca 1561 tgccattctc ctgcctcagc cttccgagta gctgggacta caggtgcccg ccaccacatc 1621 cggctaattt tttgtatttt tagtaaagat ggggtttcac catgttagcc aggatggtct 1681 cgatctcctt accttgtgat ccgcccgcct tggcctccca aagtgctggg attacaggtg 1741 tgagccacca cgcctggctg aaatacataa tcttaaaaga aaacataaga tactttattt 1801 taatatacgt gactaaatgt aaaacctaac ttattttctg ttatctattt atttttactt 1861 tcagtaacac tttttttatt ttaggtagca ttcagcctag aggcaactgc tgtttgttaa 1921 atatttcctg ttcatatatt ttgcacattt tcttatgggt tagttttctt ctcattgttt 1981 tgggaagttc ttaatatatt tggggtattt atctttcatt cgttgtctgt gtaacaaata 2041 acttctgcca tatgggttgt ctgcacattt tttggtgtct tttagtaaac aaggtttttt 2101 tgttttgtat tgttttgttt attgtaaaga tttttaaatt ttaatggagt tgatttcttt 2161 tctcattcaa gcttttgaga ataaattgga gttgaatttt t SEO ID NO: 72 Homo sapiens Mab-21 domain containing 1 (MB21D1), Human cyclic GMF1-AMP synthase isoform X1 (cGAS) amino acid sequence (XP
016865721.11 MQ PWHGKAMQ RAS EAG.ATAP KASARNARGAPMDP T E S PAAP EAA
L P KAGKFG PARKS G S RQKK SAP DTQE R P PVRAT GARAKKAPQ RAQDT Q P S DAT SAP GA
EGLEPPAAREPALSRAGSCRQRGARCSTKPRPPPGPWDVPSPGLPVSAPILVRRDAAP
GAS KLRAVLEKL KL SRDDI STAAGMVKGVVDHLLLRLKCDSAFRGVGLLNTGSYYEHV
KI SAPNEFDVMFKLEVPRI QLEEYSNTRAYYFVKFKRNP KENP L SQ FLEGEI LSAS KM
LSKFRKIIKEEINDIKDTDVIMKRKRGGSPAVTLLISEKISVDITLALESKSSWPAST
QEGLRIQNWLSAKVRKQLRLKPFYLVPKHAKEGNGFQEETWRLSFSHIEKEILNNHGK
SKTCCENKEEKCCRKDCLKLMKYLLEQLKERFKDKKHLDKFSSYHVKTAFFHMESRSV
AQAGVQWHDLGSLQALLPGFMPFSCLSLPSSWDYRCPPPHPANFLYF
SEO ID NO: 73 Peptoclostridium ditficile 630, complete genome-DisA
DNA sequence (NCBI Reference Sequence: NC 009089.1: gi1126697566:46917-47987) AT GGAGAATT TT CTAGATAATAAAAATATGCTATAT G CAT TAAAAAT GATAT CT CCT GGAACT
CCACT TA
GATTAG GT CTAAACAAT GTACTAAGAGC TAAGAC T GGT GGAT TAATT GTAAT T GCAACAAAC
GAAGAT GT
AAT GAAAATAGTAGAT GGAGGATT T G C TATAAAT GCAGAATAT T CAC CAT CATAT C TATAT
GAATTAGCT
AAAAT GGATGGAGCTATAGTTT TAAGT GGT GAT GTAAAGAAAATATTATTT GCTAATGCACAACTTATAC
CT GACTAT TT TATAGAAACAT CA.GAGACAGGAACAAGACATAGAACAGCAGAAAGAGTAGCAAPACAAAC
TGGTGCTATAGTCATAGGAATTTCACAAAGAAGAAATGTTATAACAGTTTATAGAGGAAATGAGAAGTAT
GTAGTCGAAGATATATCTAAGATATTTACTAAGGCAAATCAGGCTATACAAACTCTGGAAAAATATAAGA
CAGTATTGGACCAAGCTGTAACAAATTTAAATGCCTTAGAGTTTAATGATTTGGTAACTATTTATGATGT
TGCATTAGTCAT GCAAAAGAT GGAAAT GGTAAT GAGAGT TACAAGTATAAT T GAAAAATAT GT
GATAGAA
TT G GGT GATGAAGGAACT TTAGTAAGTATGCAAT TAGAAGAAT TAAT GG GTACAAC CAGAATAGAC
GAGA
AATTAATATTCAAAGATTATAATAAAGAAAACACAGAAATAAAAGAACTTATGAAAAAGGTCAAAAATTT
AAATTCAGAAGAACTAATAGAATTGGTTAATATGGCAAAACTATTAGGGTATAGTGGTTTTTCAGAAAGT
AT G GATAT GC CTATAAAAACAAGAGGT TATAGGAT T CT TAGCPAPAT
ACATAGACTACCAACAGCAATAA
TAGAAAACTTAGTAAATTATTTTGAAAACTTTCAACAAATTTTAGATGCATCTATTGAAGAATTAGATGA
GGT T GAAGGAATAG GT GAAATAAGAGCAACATATATAAAAAAT G GACT CATAAAAAT
GAAAC.AATTAGTC
TTATTAGATAGACACATAT GA
SEO ID NO: 74 DNA integrity scanning protein DisA [Bacillus subtilis] DNA
sequence (GenBank: KIX80328.1) atggaaaaag agaaaaaagg ggcgaaacac gagttagacc tgtcatctat attgcagttt gttgctccgg gtacaccgct cagagcgggg atggaaaacg tcttgagagc aaatacaggc ggtctgattg ttgttggata taatgataaa gtaaaagaag tggtggacgg cggctttcac ataaacacgg ctttttctcc ggcgcattta tatgagctgg ctaaaatgga tggagcgatc attttaagtg attctggtca aaagatccta tacgcgaata ctcagctgat gccggatgcc acaatttctt catcagaaac aggaatgcgg cacagaactg ccgaaagagt agctaagcaa actggctgtc ttgtaatcgc catttctgaa agaagaaatg tcataacgtt atatcaggaa aacatgaagt atacactaaa agacatagga tttattttaa ccaaggcgaa ccaagccatt caaacacttg aaaaatataa gacaatcctc gataaaacga ttaatgcact gaacgcgtta gagtttgagg aacttgttac cttcagtgat gtcttgtctg tcatgcatcg ttatgaaatg gtactgagaa tcaaaaacga aattaatatg tatatcaaag agctggggac agaagggcat ctgatcaaac tgcaagtcat tgaattgatt acggatatgg aagaagaggc cgctttattt attaaggact atgtaaaaga aaagattaaa gatccgtttg ttctcttgaa ggagctgcag gatatgtcca gttatgatct gctggatgat tccattgtgt ataagcttct cggttaccct gcttctacta atcttgatga ttatgtattg ccgagaggat acaggctgtt aaataagata ccgcgtcttc cgatgccgat tgttgaaaat gttgtagaag catttggagt cctgccaagg attattgagg cgagtgcaga agaattagat gaagtagagg gaatcggtga agtacgagcc caaaaaatca aaaaaggatt aaaacgcctg caagagaagc attatttaga cagacaactg tga SEQ ID NO: 75 DNA integrity scanning protein DisA [Bacillus subtilisl amino acid sequence fUniProtKB: sp IP37573IDISA BAC SU) SEQ ID NO: 76 response regulator receiver modulated diguanylate cyclase [Pelobacter propionicus DSM 23791 amino acid sequence (GenBank: ABK98996.1) SEQ ID NO: 77 response regulator receiver modulated diguanylate cvclase [Geobacter uraniireducens Rf4] amino acid sequence (GenBank: ABQ26076.1) SEQ ID NO: 78 response receiver-modulated diguanylate cvclase [Geobacter daltonii FRC-32] amino acid sequence (GenBank: ACM20971.1) Included in Table 1 are variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides or amino acids on the 5' end, on the 3' end, or on both the 5' and 3' ends, of the domain sequences as long as the sequence variations maintain the recited function and/or homology Included in Table 1 are nucleic acid or polypeptide molecules comprising, consisting essentially of, or consisting of:
1) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with a nucleic acid or amino acid sequence of SEQ ID NO:
1-78, or a biologically active fragment thereof;
2) a nucleic acid or amino acid sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or more nucleotides or amino acids, or any range in between, inclusive such as between 110 and 300 nucleotides or amino acids;
3) a biologically active fragment of a nucleic acid or amino acid sequence of SEQ ID NO:
1-78 having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2625, or more nucleotides or amino acids, or any range in between, inclusive such as between 110 and 300 nucleotides or amino acids;
4) a biologically active fragment of a nucleic acid or amino acid sequence of SEQ ID NO:
1-78 having 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2625, or fewer nucleotides or amino acids, or any range in between, inclusive such as between 110 and 300 nucleotides or amino acids;
II. Compositions of Matter - cyclic di-nucleotide synthetase enzyme containing Vectors, Pharmaceutical Compositions, Vaccine, Adjuvants Novel cyclic di-nucleotide synthetase enzyme containing compositions are provided herein. Such compositions (e.g., vectors, pharmaceutical compositions, adjuvants, vaccines)) are useful for the prevention and treatment of diseases, conditions, or disorders, for which an upregulation of an immune response would be beneficial. For example, the compositions may be used in the prevention or treatment of pathogenic infections, such as viral, protozoal, fungal, or bacterial infections, or cancers. Such compositions comprise any cyclic di-nucleotide synthetase enzyme (e.g., one or more DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, or any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof. In some embodiments, the compositions are provided alone or in combined with antigens (e.g., epitopes, tumor-associated antigens, or pathogen associated antigens) to enhance, stimulate, and/or increase an immune response.
In one embodiment, the DGC comprise any sequences that encode GGDEF
domains belonging to the COG2199 protein domain family, or fragment thereof.
As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (i.e., cDNA
or genomic DNA) and RNA molecules (i.e., mRNA) and analogs of the DNA or RNA
generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecules corresponding to the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (i.e., bacterial strain, V.
cholera strain). Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

A cyclic di-nucleotide synthetase enzyme nucleic acid molecule of the present invention, e.g., a nucleic acid molecule comprising the nucleotide sequence of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the C0G2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or a nucleotide sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more (e.g., about 98%) homologous to the nucleotide sequence of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or a portion thereof (i.e., 100, 200, 300, 400, 450, 500, or more nucleotides), can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a human cDNA can be isolated from a human cell line (from Stratagene, La Jolla, CA, or Clontech, Palo Alto, CA) using all or portion of the nucleic acid molecule, or fragment thereof, as a hybridization probe and standard hybridization techniques (i.e., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
Moreover, a nucleic acid molecule encompassing all or a portion of the nucleotide sequence of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or a nucleotide sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more homologous to the nucleotide sequence, or fragment thereof, can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon the sequence of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the C0G2199 protein domain family) listed herein, the Figures, and the Example, or a biologically active fragment thereof, or the homologous nucleotide sequence.
For example, mRNA can be isolated from cells of interest and cDNA can be prepared using reverse transcriptase (i.e., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St.
Petersburg, FL). Synthetic oligonucleotide primers for PCR amplification can be designed according to well-known methods in the art. A nucleic acid of the present invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA
sequence analysis. Furthermore, oligonucleotides corresponding to the nucleotide sequence of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, can be prepared by standard synthetic techniques, i.e., using an automated DNA
synthesizer.
Probes based on the nucleotide sequences of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, can be used to detect transcripts or genomic sequences encoding the same or homologous sequences. In some embodiments, the probe further comprises a label group attached thereto, e., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which express one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncVDisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, such as by measuring a level of nucleic acid in a sample of cells from a subject, i.e., detecting mRNA levels of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof.
Nucleic acid molecules corresponding to one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, from different species are also contemplated. In one embodiment, the nucleic acid molecule(s) of the present invention encodes a cyclic di-nucleotide synthetase enzyme or portion thereof which includes a nucleic acid sequence sufficiently similar to the nucleic acid sequence of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, such that the enzyme or portion thereof has enzymatic activity as described herein. Such homologous nucleic acids and encoded polypeptides can be readily produced by the ordinarily skilled artisan based on the sequence information provided herein, the Figures, and the Examples.
As used herein, the language "sufficiently homologous" refers to nucleic acids or portions thereof which have nucleic acid sequences which include a minimum number of identical or equivalent (e.g., a cognate pair of nucleotides for maintaining nucleic acid secondary structure) to a nucleic acid sequence of the cyclic di-nucleotide synthetase enzyme, or fragment thereof, such that the nucleic acid thereof modulates (e.g, enhances) one or more of the following biological activities: a) increase c-di-GMP, c-di-AMP, cGAMP, and/or any cyclic di-nucleotide; b) enhance innate immue response; c) stimulate adaptive immune response; and d) increase humoral immune response.
Portions of nucleic acid molecules of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, are preferably biologically active portions of the protein. As used herein, the term "biologically active portion" of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, is intended to include a portion, e.g., a domain/motif, that has one or more of the biological activities of the full-length protein.
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-G-GDEFs, DncV, DisA, cGAS any sequences that encode GGDEF
domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or fragment thereof due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence, or fragment thereof. In another embodiment, an isolated nucleic acid molecule of the present invention has a nucleotide sequence having a nucleic acid sequence of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or fragment thereof, or having a nucleic acid sequence which is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or fragment thereof. In another embodiment, a nucleic acid encoding a polypeptide consists of nucleic acid sequence encoding a portion of a full-length fragment of interest that is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or more nucleotides, or any range in between, inclusive such as between 110 and 300 nucleotides; or more nucleotides, or any range in between, inclusive such as between 110 and 300 nucleotides; or 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2625, or fewer nucleotides, or any range in between, inclusive such as between 110 and 300 nucleotides.
It will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, may exist within a population.
Such genetic polymorphisms may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, preferably bacterial, e.g., V. cholerae DGC. Such natural allelic variations can typically result in 1-5%
variance in the nucleotide sequence of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof. Any and all such nucleotide variations and resulting amino acid polymorphisms in the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, that are the result of natural allelic variation and that do not alter the functional activity of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, are intended to be within the scope of the present invention. Moreover, nucleic acid molecules encoding one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, from other species.
In addition to naturally-occurring allelic variants of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence, or fragment thereof, thereby leading to changes in the amino acid sequence of the encoded one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, without altering the functional ability of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof For example, nucleotide substitutions leading to substitutions at "non-essential" nucleotide positions can be made in the sequence, or fragment thereof. A "non-essential" amino acid position is a position that can be altered from the wild-type sequence of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, without substantially altering the activity of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, whereas an "essential" amino acid residue is required for the activity of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof. Other positions, however, (e.g., those that are not conserved or only semi-conserved between mouse and human) may not be essential for activity and thus are likely to be amenable to alteration without altering the activity of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof.
The term "sequence identity or homology" refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology. Unless otherwise specified "loop out regions", e.g., those arising from, from deletions or insertions in one of the sequences are counted as mismatches.
The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. Preferably, the alignment can be performed using the Clustal Method. Multiple alignment parameters include GAP
Penalty =10, Gap Length Penalty = 10. For DNA alignments, the pairwise alignment parameters can be Htuple=2, Gap penalty=5, Window=4, and Diagonal saved=4. For protein alignments, the pairwise alignment parameters can be Ktuple=1, Gap penalty=3, Windovv=5, and Diagonals Saved=5.
In some embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available online), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available online), using a NWSgapdna.CMP
matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0) (available online), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
An isolated nucleic acid molecule encoding a protein homologous to one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or fragment thereof, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence, or fragment thereof, or a homologous nucleotide sequence such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
The levels of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, levels may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed molecule or protein.
Non-limiting examples of such methods include immunological methods for detection of proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.
In some embodiments, the levels of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, levels are ascertained by measuring gene transcript (e.g., mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity. Expression levels can be monitored in a variety of ways, including by detecting cyclic di-nucleotide synthetase enzyme levels or activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, transcribed RNA, cyclic di-nucleotide synthetase enzyme activity), or, alternatively, can be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.
In a particular embodiment, the RNA expression level can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art The term "biological sample" is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject.
Many expression detection methods use isolated RNA. For in vitro methods, any RNA
isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Patent No. 4,843,155).
The isolated RNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One diagnostic method for the detection of RNA levels involves contacting the isolated RNA with a nucleic acid molecule (probe) that can hybridize to the RNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an RNA or genomic DNA encoding one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof. Other suitable probes for use in the diagnostic assays of the present invention are described herein.
Hybridization of an RNA with the probe indicates that one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the C0G2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, is being expressed.
In one format, the RNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated RNA on an agarose gel and transferring the RNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the RNA is contacted with the probe(s), for example, in a gene chip array, e.g., an AffymetrixTM gene chip array. A
skilled artisan can readily adapt known RNA detection methods for use in detecting the level of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, RNA expression levels An alternative method for determining RNA expression level in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sc!. USA, 88:189-193), self-sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sc!. USA 87:1874-1878), transcriptional amplification system (Kwoh etal., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal., 1988, Bio/Technology 6:1197), rolling circle replication (Lizardi et al.,U U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
For in situ methods, RNA does not need to be isolated from the cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof.
As an alternative to making determinations based on the absolute expression level, determinations may be based on the normalized expression level of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof.
Expression levels are normalized by correcting the absolute expression level by comparing its expression to the expression of a non-cyclic di-nucleotide synthetase enzyme gene, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes This normalization allows the comparison of the expression level in one sample, e.g., a subject sample, to another sample, e.g., a normal sample, or between samples from different sources.
The level or activity of a protein corresponding to one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, can also be detected and/or quantified by detecting or quantifying the activity, such as effects on associate polypeptides like transcription factors or nuclear receptors. The associated polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. These may include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, liquid chromatrography tandem mass spectrometry (LC-MS/MS) and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, and the like. A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells express the cyclic di-nucleotide synthetase enzyme of interest.
a. Cyclic di-nucleotide synthetase enzyme gene containing Vectors In some embodiments, vectors and/or host cells are further provided. One aspect of the present invention pertains to the use of recombinant vectors (e.g., gene therapy vectors), containing a nucleic acid encoding a cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or a portion or ortholog thereof As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of recombinant vectors (e.g., viral vectors, replication defective adenoviruses, any human or non-human adenovirus, AAV, DNA-based vector, retroviruses, or lentiviruses), which serve equivalent functions. In one embodiment, vectors comprising a cyclic di-nucleotide synthetase enzyme nucleic acid molecule are used.
The recombinant vectors (e.g., gene thereapy vectors) of the present invention comprise any of the nucleic acid encoding a cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF
domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or a portion or ortholog thereof, in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
Within a recombinant vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the recombinant vector (e.g., gene therapy vector) can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The recombinant vectors (e.g., gene therapy vectors) of the present invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
The recombinant vectors (e.g., gene therapy vectors) of the present invention comprising any of the nucleic acid encoding a cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF

domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or a portion or ortholog thereof, can be designed for expression of the desired cyclic di-nucleotide synthetase enzyme in prokaryotic or eukaryotic cells. For example, a cyclic di-nucleotide synthetase enzyme can be expressed in bacterial cells such as E. coil, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA
(1990). Alternatively, the recombinant vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Examples of suitable inducible non-fusion E. coli vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET lid (Studier etal., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). Examples of suitable yeast vectors include pYepSecl (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Examples of suitable baculovirus vectors useful for insect cell hosts include the pAc series (Smith etal.
(1983)Mol Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
Examples of suitable mammalian vectors include CMV-containing vectors, such as pCDM8 (Seed, B. (1987) Nature 329:840), and pMT2PC (Kaufman etal. (1987)1MB0J 6:187-195).
In another embodiment, the recombinant vector (e.g., gene theray vector) comprising any of the nucleic acid encoding a cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF
domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or a portion or ortholog thereof, is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters such as in melanoma cancer cells are well-known in the art (see, for example, Pleshkan et al. (2011) Acta Nat. 3:13-21).
The present invention further provides a recombinant vector (e.g., gene therapy vector) comprising any of the nucleic acid encoding a cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or a portion or ortholog thereof, cloned into the recombinant vector (e.g., gene therapy vector) in an antisense orientation.
That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA
molecule which is antisense to a cyclic di-nucleotide synthetase enzyme mRNA described herein.
Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
Another aspect of the present invention pertains to host cells into which a recombinant vector comprising any of the nucleic acid encoding a cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or a portion or ortholog thereof has been introduced. The terms "host cell" and "recombinant host cell"
are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, cyclic di-nucleotide synthetase enzyme protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Fao hepatoma cells, primary hepatocytes, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, etal. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. A cyclic di-nucleotide synthetase enzyme polypeptide or fragment thereof, may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, a cyclic di-nucleotide synthetase enzyme polypeptide or fragment thereof, may be retained cytoplasmically and the cells harvested, lysed and the protein or protein complex isolated. A cyclic di-nucleotide synthetase enzyme polypeptide or fragment thereof, may be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and inmmunoaffinity purification with antibodies specific for particular epitopes of a cyclic di-nucleotide synthetase enzyme or a fragment thereof. In other embodiments, heterologous tags can be used for purification purposes (e.g., epitope tags and FC fusion tags), according to standards methods known in the art.
Thus, a nucleotide sequence encoding all or a selected portion of a cyclic di-nucleotide synthetase enzyme polypeptide may be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes. Ligating the sequence into a polynucleotide construct, such as an recombinant vector (e.g., gene therapy vector), and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures. Similar procedures, or modifications thereof, may be employed to prepare recombinant cyclic di-nucleotide synthetase enzyme polypeptides, or fragments thereof, by microbial means or tissue-culture technology in accord with the subject invention.
A host cell of the present invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) cyclic di-nucleotide synthetase enzyme protein. Accordingly, the invention further provides methods for producing cyclic di-nucleotide synthetase enzyme protein using the host cells of the present invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant vector encoding a cyclic di-nucleotide synthetase enzyme has been introduced) in a suitable medium until cyclic di-nucleotide synthetase enzyme protein is produced. In another embodiment, the method further comprises isolating the cyclic di-nucleotide synthetase enzyme portein from the medium or the host cell.
The host cells of the present invention can also be used to produce nonhuman transgenic animals. The nonhuman transgenic animals can be used in screening assays designed to identify compositions or compounds, e.g., drugs, pharmaceuticals, etc., which are capable of modulation (e.g., upregulating) an immune response. For example, in one embodiment, a host cell of the present invention is a fertilized oocyte or an embryonic stem cell into which cyclic di-nucleotide synthetase enzyme encoding sequences, or fragments thereof, have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous cyclic di-nucleotide synthetase enzyme sequences have been introduced into their genome or homologous recombinant animals in which endogenous cyclic di-nucleotide synthetase enzyme sequences have been altered.
Such animals are useful for studying the function and/or activity of cyclic di-nucleotide synthetase enzyme, or fragments thereof, and for identifying and/or evaluating modulators of cyclic di-nucleotide synthetase enzyme activity. As used herein, a "transgenic animal" is a nonhuman animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include nonhuman primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a nonhuman animal, preferably a mammal, more preferably a mouse, in which an endogenous cyclic di-nucleotide synthetase enzyme gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the present invention can be created by introducing nucleic acids encoding a cyclic di-nucleotide synthetase enzyme, or a fragment thereof, into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Human cyclic di-nucleotide synthetase enzyme cDNA sequence can be introduced as a transgene into the genome of a nonhuman animal. Alternatively, a nonhuman homologue of the human cyclic di-nucleotide synthetase enzyme gene can be used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the cyclic di-nucleotide synthetase enzyme transgene to direct expression of cyclic di-nucleotide synthetase enzyme protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, both by Leder et al.,U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the cyclic di-nucleotide synthetase enzyme transgene in its genome and/or expression of cyclic di-nucleotide synthetase enzyme mRNA in tissues or cells of the animals. A
transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a cyclic di-nucleotide synthetase enzyme can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of cyclic di-nucleotide synthetase enzyme gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the cyclic di-nucleotide synthetase enzyme gene. The cyclic di-nucleotide synthetase enzyme gene can be a bacterial gene. The cyclic di-nucleotide synthetase enzyme gene can be a human gene, but more preferably, is a non-human homologue of a human cyclic di-nucleotide synthetase enzyme gene. For example, a mouse cyclic di-nucleotide synthetase enzyme gene can be used to construct a homologous recombination vector suitable for altering an endogenous cyclic di-nucleotide synthetase enzyme gene, respectively, in the mouse genome. In another embodiment, the vector is designed such that, upon homologous recombination, the endogenous cyclic di-nucleotide synthetase enzyme gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out"
vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous DGC gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous cyclic di-nucleotide synthetase enzyme protein).
In the homologous recombination vector, the altered portion of the cyclic di-nucleotide synthetase enzyme gene is flanked at its 5' and 3' ends by additional nucleic acid of the cyclic di-nucleotide synthetase enzyme gene to allow for homologous recombination to occur between the exogenous cyclic di-nucleotide synthetase enzyme gene carried by the vector and an endogenous cyclic di-nucleotide synthetase enzyme gene in an embryonic stem cell.
The additional flanking cyclic di-nucleotide synthetase enzyme gene nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced cyclic di-nucleotide synthetase enzyme gene has homologously recombined with the endogenous cyclic di-nucleotide synthetase enzyme gene are selected (see e.g., Li, E. etal. (1992) Cell 69:915).
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A
chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO
91/01140 by Smithies etal.; WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et al.
In another embodiment, transgenic nonhuman animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage Pl. Fora description of the crelloxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl.
Acad Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP

recombinase system of Saccharomyces cerevisiae (O'Gorman etal. (1991) Science 251:1351-1355. If a creilorP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double"
transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the nonhuman transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated Nucleic acid molecules of the present invention can also be engineered as fusion constructs using recombinant DNA techniques. A "chimeric cyclic di-nucleotide synthetase enzyme" or "fusion cyclic di-nucleotide synthetase enzyme"
comprises a cyclic di-nucleotide synthetase enzyme polypeptide described herein operatively linked to a non-cyclic di-nucleotide synthetase enzyme nucleic acid sequence. Within the fusion construct, the term "operatively linked" is intended to indicate that the cyclic di-nucleotide synthetase enzyme nucleic acid sequence and the non- cyclic di-nucleotide synthetase enzyme nucleic acid sequence are fused in a rame to each other. The cyclic di-nucleotide synthetase enzyme polypeptide can be fused to the 5' end, the 3' end, or in between the 5' and 3' ends of the cyclic di-nucleotide synthetase enzyme nucleic acid sequence. The fusion protein can function as a nucleic acid (e.g., a MS2 loop structure) or encode a protein for translation, such as using an internal ribosome entry sequence (IRES). For example, in one embodiment the fusion protein is a cyclic di-nucleotide synthetase enzyme -GST
and/or cyclic di-nucleotide synthetase enzyme -Fe fusion protein. Such fusion proteins can facilitate the purification, expression, and/or bioavailability of recombinant cyclic di-nucleotide synthetase enzyme constructs. In certain host cells (e.g., mammalian host cells), expression and/or secretion of the cyclic di-nucleotide synthetase enzyme fusion construct can be increased through use of a heterologous signal sequence.

Preferably, a cyclic di-nucleotide synthetase enzyme chimeric or fusion constructs (e.g., gene therapy vectors comprising cyclic di-nucleotide synthetase enzyme) of the present invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different sequences are ligated together in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons:
1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A cyclic di-nucleotide synthetase enzyme-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the cyclic di-nucleotide synthetase enzyme protein.
Systematic substitution of one or more amino acids of a polypeptide amino acid sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. In addition, constrained peptides comprising a polypeptide amino acid sequence of interest or a substantially identical sequence variation can be generated by methods known in the art (Rizo and Gierasch (1992) Annu.
Rev.
Biochem. 61:387, incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
The amino acid sequences disclosed herein will enable those of skill in the art to produce polypeptides corresponding peptide sequences and sequence variants thereof.
Such polypeptides can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding the peptide sequence, frequently as part of a larger polypeptide.
Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous proteins in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are described further in Maniatis et al. Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.;
Merrifield, J. (1969)1 Am. Chem. Soc. 91:501; Chaiken I. M. (1981) CRC Crit. Rev. Biochem.
11:
255; Kaiser et a/. (1989) Science 243:187; Merrifield, B. (1986) Science 232:342; Kent, S.
B. H. (1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which are incorporated herein by reference).
Peptides can be produced, typically by direct chemical synthesis. Peptides can be produced as modified peptides, with nonpeptide moieties attached by covalent linkage to the N-terminus and/or C-terminus. In certain embodiments, either the carboxy-terminus or the amino-terminus, or both, are chemically modified. The most common modifications of the terminal amino and carboxyl groups are acetylation and amidation, respectively.
Amino-terminal modifications such as acylation (e.g., acetylation) or alkylation (e.g., methylation) and carboxy-terminal-modifications such as amidation, as well as other terminal modifications, including cyclization, can be incorporated into various embodiments of the present invention. Certain amino-terminal and/or carboxy-terminal modifications and/or peptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties, such as:
enhanced stability, increased potency and/or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others. Peptides disclosed herein can be used therapeutically to treat disease.
b. Pharmaceutical compositions, adjuvants, vaccines In another aspect, the present invention provides pharmaceutically acceptable compositions, adjuvants, and vaccines which comprise a therapeutically-effective amount of a recombinant vector (e.g., gene therapy vector comprising any of the nucleotide sequence of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or fragment thereof) which increases cyclic di-nucleotide synthetase enzyme, c-di-GMP, c-di-AMP, c-di-GAMP, any cyclic di-nucleotide, or immune response levels and/or activity, formulated together with one or more pharmaceuticallyacceptable carriers (additives) and/or diluents. In some embodiments, the pharmaceutical compositions, adjuvants, and vaccines comprises a first gene therapy vector (e.g., gene therapy vector containing any of the nucleotide sequence of the one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or fragment thereof) in combination with a extracellular antigen, epitope, or peptide (naked or provided in an gene therapy vector). In some embodiments, the pharmaceutical compositions, adjuvants, and vaccines can be combined with any immune modulating, anti-viral, anti-bacterial, anti-cancer, chemotherapeutic, or immunotherapeutic compositions.
Immunotherapeutic compositions, include, but are not limited to, ipilimumab (Yervoye), trastuzumab (Herceptine), rituximab (Rituxane), bevacizumab (Avastin0), pertuzumab (Omnitarg0), tositumomab (Bexxare), edrecolomab (Panorexe), and G250.
Compounds of the present invention can also be combined with, or used in combination with, anti-TNF-a antibodies. Large molecule active compositions may be administered in the form of anti-cancer vaccines. For example, compositions that secrete, or cause the secretion of, cytokines such as IL-2, G-CSF, and GM-CSF can be used in the methods, pharmaceutical compositions, and kits provided herein. See, e.g., Emens, L.
A., et al., Cliff.
Opinion Mol. Ther. 3(1):77-84 (2001).
Second active compositions that are small molecules can also be used to in combination with the compositions of the present invention. Examples of small molecule second active compositions include, but are not limited to, anti-cancer compositions, antibiotics, antivirals, immunosuppressive compositions, and steroids.
In some embodiments, well known "combination chemotherapy" regimens can be used. In one embodiment, the combination chemotherapy comprises a combination of two or more of cyclophosphamide, hydroxydaunorubicin (also known as doxorubicin or adriamycin), oncovorin (vincristine), and prednisone. In another embodiment, the combination chemotherapy comprises a combination of cyclophsophamide, oncovorin, prednisone, and one or more chemotherapeutics selected from the group consisting of anthracycline, hydroxydaunorubicin, epirubicin, and motixantrone.
Examples of other anti-cancer compositions include, but are not limited to:
acivicin;
aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;
altretamine;
ambomycin; ametantrone acetate; amsacrine; anastrozole; anthramycin;
asparaginase;
asperlin, azacitidine; azetepa, azotomycin, batimastat; benzodepa;
bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium;
bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer;
carboplatin;

carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor);
chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;
cyclophosphamide;
cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;

dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;
doxorubicin;
doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate;
duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin;
enpromate;
epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;
estramustine;
estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate;
etoprine;
fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate;
fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine;
gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine;
iproplatin;
irinotecan; irinotecan hydrochloride; lanreotide acetate; letrozole;
leuprolide acetate;
liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride;
masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate;
melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;
metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;
mitomalcin;
mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid;
nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase;
peliomycin;
pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;
piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin;
prednimustine;
procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin;
riboprine;
safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium;
sparsomycin;
spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin;
streptozocin;
sulofenur; talisomycin; tecogalan sodium; taxotere; tegafur; teloxantrone hydrochloride;
temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine;
thiotepa;
tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate;
trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride;
uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;
vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;
zinostatin; and zorubicin hydrochloride.
Other anti-cancer drugs include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene;
adecypenol;

adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox;

amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide;
anastrozole;
andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;
antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma;
antiestrogen;
antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase;
asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;
azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL
antagonists;
benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine;
betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene;
bisaziridinylspermine;
bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;
buthionine sulfoximine;
calcipotriol; calphostin C; camptothecin derivatives; capecitabine;
carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;
carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B;
cetrorelix;
chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;
clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4;
combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A
derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cyclosporin A;
cypemycin;
cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine;
dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone;
didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-;
dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron;
doxifluridine;
doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;
edelfosine;
edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride;
estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate;
exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;
flavopiridol;
flezelastine; fluasterone; fludarabine; fluorodaunoninicin hydrochloride;
forfenimex;
formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine;
ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors;
hepsulfam; heregulin;
hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;

idramantone; ilmofosine; ilomastat; imatinib (e.g., Gleevece), imiquimod;
immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor;
interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;

irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F;
lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin;
letrozole; leukemia inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds;
lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; loxoribine;
lurtotecan;
lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;
marimastat;
masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril;
merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor;
mifepristone;
miltefosine; mirimostim; mitoguazone; mitolactol; mitomycin analogues;
mitonafide;
mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene;
molgramostim;
Erbitux, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; mustard anticancer composition; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin;
nagrestip;
naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;
nemorubicin;
neridronic acid; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant;
nitrullyn; oblimersen (Genasenseg); 06-benzylguanine; octreotide; okicenone;
oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer;
ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;
paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;
panaxytriol;
panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide, perillyl alcohol;
phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex;
porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein lcinase C inhibitor;
protein kinase C
inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; MI retinamide; rohituldne; romurtide;
roquinimex;
rubiginone BI; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A;
sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides;
signal transduction inhibitors; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate;
solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D;

spiromustine, splenopentin, spongistatin 1; squalamine; stipiamide; stromely sin inhibitors;
sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista;
suramin;
swainsonine; tallimustine; tamoxifen methiodide; tauromustine; tazarotene;
tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;
teniposide;
tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline, thrombopoietin;
thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist;
thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin;
toremifene; translation inhibitors; tretinoin; triacetyluridine; triciribine;
trimetrexate;
triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;
tyrphostins; UBC inhibitors;
ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; velaresol; veramine; verdins;
verteporfin; vinorelbine;
vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
Specific second active compositions include, but are not limited to, chlorambucil, fludarabine, dexamethasone (Decadrong), hydrocortisone, methylprednisolone, cilostamide, doxorubicin (Doxi10), forskolin, rituximab, cyclosporin A, cisplatin, vincristine, PDE7 inhibitors such as BRL-50481 and IR-202, dual PDE4/7 inhibitors such as IR-284, cilostazol, meribendan, milrinone, vesnarionone, enoximone and pimobendan, Syk inhibitors such as fostamatinib di sodium (R406/R788), R343, R-112 and Excellair (ZaBeCor Pharmaceuticals, Bala Cynwyd, Pa.).
Antiviral, antifungal, and/or antibacterial compositions, include but not limited, cidofovir and interleukin-2, Cytarabine (also known as ARA-C), isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, kanamycin, amikacin, capreomycin, ofloxacin, levofioxacin, moxifioxacin, cycloserine, para-aminosaicylic acid, ethioamide, prothionamide, thioacetazone, clofazimine, amoxicilin with clavulanate, imipenem, linezolid, clarithromycin, thioridazine, bicyclic nitroimidazoles (e.g., (S)-6,7-dihydro-2-nitro-64[4-(trifluoromethoxy)phenyl]methoxy1-5H-imidazo[2,1 ,Thxazine (PA-824) and TBA-354, available from TB Alliance), bedaquiline (TMC-207), delamanid (0PC67683), oxazolidinone, 2-[(2S)-2- methyl-1 ,4-dioxa-8-azaspiro[4.5]decan-8-y1]-8-nitro-6-trifluoromethy1-4H-1,3- benzothiazin-4-one (BTZ043), imidazopyridines (e.g. ,Q201 , available from Quro Science Inc.), anti-interleukin 4 neutralizing antibodies, high-dose intravenous immunoglobulin, 16a- bromoepiandosterone (HE2000), RUTI vaccine, DNA
vaccine with HSP65, Ag85, MPT-64, and MPT-83, dzherelo (plant extracts from the Ukraine), cytokines (such as Interleukin 2, Interleukin 7, Interleukin 15, Interleukin 27, Interleukin 12, Interferon y, corticosteroids, thalidomide, etanercept, steroids, prednisone, (NNRTIs), such as efavirenz (Sustiva), etravirine (Intelence) and nevirapine (Viramune);
Nucleoside reverse transcriptase inhibitors (NRTIs), such as Abacavir (Ziagen), and the combination drugs emtricitabine and tenofovir (Truvada), and lamivudine and zidovudine (Combivir); Protease inhibitors (Pis), such as atazanavir (Reyataz), darunavir (Prezista), fosamprenavir (Lexiva) and ritonavir (Norvir); Entry or fusion inhibitors, such enfuvirtide (Fuzeon) and maraviroc (Selzentry); and Integrase inhibitors, such as Raltegravir (Isentress).
As described in detail below, the pharmaceutical compositions, adjuvants, and vaccines of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension;
(3) topical application, for example, as a cream, ointment or spray applied to the skin;
(4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
The phrase "therapeutically-effective amount" as used herein means that amount of a composition of matter of the present invention that modulates immune response levels and/or activity, which is effective for producing some desired therapeutic effect, e.g., pathogenic infection or cancer treatment, at a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable" is employed herein to refer to those pharmaceutical compositions, adjuvants, vaccines, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering compositions, such as magnesium hydroxide and aluminum hydroxide;
(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
Formulations useful in the methods of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1% to about 99% of active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.
Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an composition as an active ingredient. A compound may also be administered as a bolus, electuary or paste.
In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating compositions, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding compositions, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting compositions, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring compositions. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering compositions.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing composition. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.
Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing compositions in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
These compositions may also optionally contain opacifying compositions and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
Examples of embedding compositions, which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing compositions and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting compositions, emulsifying and suspending compositions, sweetening, flavoring, coloring, perfuming and preservative compositions.
Suspensions, in addition to the active composition may contain suspending compositions as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more therapeutic compositions with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active composition.
Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate Dosage forms for the topical or transdermal administration of an composition that modulates (e.g., increases) immune response levels and/or activity include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
The active component may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to a therapeutic composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to an composition that modulates (e.g., increases) immune response levels and/or activity, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
The composition that modulates (e.g., increases) immune response levels and/or activity, can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A
nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the composition to shear, which can result in degradation of the compound.
Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the composition together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
Transdermal patches have the added advantage of providing controlled delivery of a therapeutic composition to the body. Such dosage forms can be made by dissolving or dispersing the composition in the proper medium. Absorption enhancers can also be used to increase the flux of the peptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the peptidomimetic in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more therapeutic compositions in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening compositions.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting compositions, emulsifying compositions and dispersing compositions. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal compositions, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic compositions, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of compositions which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of an composition that modulates (e.g., increases) immune response levels and/or activity, in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.
When the compositions of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be determined by themethods of the present invention so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
The cyclic di-nucleotide synthetase enzyme containing vectors can be used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054 3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., adenoviralviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
III. Uses and Methods of the present invention The compositions of matter of the present invention comprising a vector (e.g., any gene therapy vector compring the nucleotide sequence of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof or a portion thereof) can be used in one or more of the following methods: a) method of inducing or enhancing an immune response in a mammal and b) methods of treatment (e.g., therapeutic and prophylactic) in a mammal (e.g., human) having a condition that would benefit from upregulation of an immune response.
In one aspect, the present invention provides a method for preventing in a subject a pathogenic infection, by administering to the subject the compositions of matter of the present invention which modulates cyclic di-nucleotide synthetase enzyme expression or at least one activity of the cyclic di-nucleotide synthetase enzyme.
Administration of such compositions can occur prior to the manifestation of symptoms characteristic of the pathogenic infection, such that an infection is prevented or, alternatively, delayed in its progression.
Another aspect of the present invention pertains to methods of modulating the expression or activity of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or fragments thereof, for therapeutic purposes.
Accordingly, the activity and/or expression of the cyclic di-nucleotide synthetase enzyme can be modulated in order to modulate the immune response.
The present invention also contemplates a method for enhancing an immune response comprising the administration to a subject the compositions of the present invention as part of a vaccination regimen. The present invention is particularly useful in pharmaceutical vaccines and genetic vaccines in humans.
Adjuvants promote the immune response in a number of ways such as to modify the activities of immune cells that are involved with generating and maintaining the immune response. Additionally, adjuvants modify the presentation of antigen to the immune system.
The compositions of the invention (e.g., the recombinant vectors (e.g., gene therapy vectors) containing at least one nucleic acid encoding a cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or a portion or ortholog thereof) may be used as adjuvants in a vaccination regimen.
Another aspect of the invention pertains to therapeutic methods of modulating an immune response, e.g., enhancing or increasing an immune response by transducing DGC
using an adenovirus to increase c-di-GMP levels.

Modulatory methods of the present invention involve contacting a cell, such as an immune cell with any of the compositions of matter (e.g., any gene therapy vector comprising the nucleotide sequence of one or more cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof or a portion thereof).
Exemplary compositions useful in such methods are described above. Such compositions can be administered in vitro or ex vivo (e.g., by contacting the cell with the composition) or, alternatively, in vivo (e.g., by administering the compositions to a subject).
As such, the present invention provides methods useful for treating an individual afflicted with a condition that would benefit from an increased immune response, such as a pathogenic infection or a cancer.
Compositions that upregulate immune responses can be in the form of enhancing an existing immune response or eliciting an initial immune response. Thus, enhancing an immune response using the subject compositions and methods is useful for treating cancer, but can also be useful for treating an infectious disease (e.g., bacteria, viruses, or parasites), a parasitic infection, and an immunosuppressive disease.
Exemplary infectious disorders include viral skin diseases, such as Herpes or shingles, in which case such a composition can be delivered topically to the skin. In addition, systemic viral diseases, such as influenza, the common cold, and encephalitis might be alleviated by systemic administration of such compositions.
Immune responses can also be enhanced in an infected patient through an ex vivo approach, for instance, by removing immune cells from the patient, contacting immune cells in vitro with an composition described herein and reintroducing the in vitro stimulated immune cells into the patient.
In certain instances, it may be desirable to further administer other compositions that upregulate immune responses. Such additional compositions and therapies are described further below.
Compositions that upregulate an immune response can be used prophylactically in vaccines against various polypeptides (e.g., polypeptides derived from pathogens).
Immunity against a pathogen (e.g., a virus) can be induced by vaccinating with a viral protein or antigen along with a recombinant vector (e.g., gene therapy vector contining cyclic di-nucleotide synthetase enzyme) as an appropriate adjuvant for upregulatingan immune response,.
In another embodiment, upregulation or enhancement of an immune response function, as described herein, is useful in the induction of tumor immunity.
In another embodiment, the immune response can be stimulated by the methods described herein, such that preexisting tolerance, clonal deletion, and/or exhaustion (e.g., T
cell exhaustion) is overcome. For example, immune responses against antigens to which a subject cannot mount a significant immune response, such as a pathogen specific or tumor specific antigens can be induced by administering appropriate compositions described herein that upregulate the immune response. In one embodiment, an extracellular antigen, such as a pathogen-specific or tumor-specific antigen, can be coadministered.
In another embodiment, the subject compositions can be used as adjuvants to boost responses to foreign antigens in the process of active immunization.
In still another embodiment, compositions described herein useful for upregulating immune responses can further be linked, or operatively attached, to toxins using techniques that are known in the art, e.g., crosslinking or via recombinant DNA
techniques. Such compositions can result in cellular destruction of desired cells. In one embodiment, a toxin can be conjugated to an antibody, such as a bispecific antibody. Such antibodies are useful for targeting a specific cell population, e.g., using a marker found only on a certain type of cell. The preparation of immunotoxins is, in general, well known in the art (see, e.g., U .S .
Pat. Nos. 4,340,535, and EP 44167). Numerous types of disulfide-bond containing linkers are known which can successfully be employed to conjugate the toxin moiety with a polypeptide. In one embodiment, linkers that contain a disulfide bond that is sterically "hindered" are preferred, due to their greater stability in vivo, thus preventing release of the toxin moiety prior to binding at the site of action. A wide variety of toxins are known that may be conjugated to polypeptides or antibodies of the invention. Examples include:
numerous useful plant-, fungus- or even bacteria-derived toxins, which, by way of example, include various A chain toxins, particularly ricin A chain, ribosome inactivating proteins such as saporin or gelonin, a-sarcin, aspergillin, restrictocin, ribonucleases, such as placental ribonuclease, angiogenic, diphtheria toxin, and Pseudomonas exotoxin, etc. A
preferred toxin moiety for use in connection with the invention is toxin A
chain which has been treated to modify or remove carbohydrate residues, deglycosylated A
chain. (U.S.

Patent 5,776,427). Infusion of one or a combination of such cytotoxic compositions, (e.g., ricin fusions) into a patient may result in the death of immune cells.
In another embodiment, certain combinations work synergistically in the treatment of conditions that would benefit from the modulation of immune responses.
Second active compositions can be large molecules (e.g., proteins) or small molecules (e.g., synthetic inorganic, organometallic, or organic molecules). For example, anti-virals or anti-cancer compositions can be further combined with the compositions of the present invention to enhance or stimulate an immune response.
In one embodiment, anti-cancer immunotherapy is administered in combination to subjects described herein. The term "immunotherapy" refers to any therapy that acts by targeting immune response modulation (e.g., induction, enhancement, suppression, or reduction of an immune response). In certain embodiments, immunotherapy is administered that ativates T cells that recognize neoantigens (e.g., mutants that change the normal protein coding sequence and can be processed by the antigen presentation system, bind to MHC and recognized as foreign by T cells).
The term "immune response" includes T cell-mediated and/or B cell-mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity. In addition, the term "immune response"
includes immune responses that are indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages. The term "inhibit" includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction. In some embodiments, cancer is "inhibited"
if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented. As used herein, cancer is also "inhibited" if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented. The term "promote" has the opposite meaning.
The term "immunotherapeutic composition" can include any molecule, peptide, antibody or other composition which can modulate a host immune system in response to an antigen, such as expressed by a tumor or cancer in the subject.
Immunotherapeutic strategies include administration of vaccines, antibodies, cytokines, chemokines, as well as small molecular inhibitors, anti-sense oligonucleotides, and gene therapy, as described further below (see, for example, Mocellin el al. (2002) Cancer Immunol.
Immunother.
51:583-595; Dy et al. (2002) J. Cl/n. Oncol. 20: 2881-2894).

Immunotherapies that are designed to elicit or amplify an immune response are referred to as "activation immunotherapies." Immunotherapies that are designed to reduce or suppress an immune response are referred to as "suppression immunotherapies." Any composition believed to have an immune system effect on the genetically modified transplanted cancer cells can be assayed to determine whether the composition is an immunotherapy and the effect that a given genetic modification has on the modulation of immune response. In some embodiments, the immunotherapy is cancer cell-specific.
Immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic composition or toxin, to a tumor antigen). Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines.
In one embodiment, immunotherapy comprises adoptive cell-based immunotherapies. Well known adoptive cell-based immunotherapeutic modalities, including, without limitation, Irradiated autologous or al logeneic tumor cells, tumor lysates or apoptotic tumor cells, antigen-presenting cell-based immunotherapy, dendritic cell-based immunotherapy, adoptive T cell transfer, adoptive CAR T cell therapy, autologous immune enhancement therapy (AIET), cancer vaccines, and/or antigen presenting cells.
Such cell-based immunotherapies can be further modified to express one or more gene products to further modulate immune responses, such as expressing cytokines like GM-CSF, and/or to express tumor-associated antigen (TAA) antigens, such as Mage-1, gp-100, patient-specific neoantigen vaccines, and the like.
In another embodiment, immunotherapy comprises non-cell-based immunotherapies. In one embodiment, compositions comprising antigens with or without vaccine-enhancing adjuvants are used Such compositions exist in many well known forms, such as peptide compositions, oncolytic viruses, recombinant antigen comprising fusion proteins, and the like. In still another embodiment, immunomodulatory interleukins, such as IL-2, IL-6, IL-12, IL-17, IL-23, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In yet another embodiment, immunomodulatory cytokines, such as interferons, G-CSF, imiquimod, TNFalpha, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In another embodiment, immunomodulatory chemokines, such as CCL3, CCL26, and CXCL7, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In another embodiment, immunomodulatory molecules targeting immunosuppression, such as STAT3 signaling modulators, NFkappaB signaling modulators, and immune checkpoint modulators, are used. The terms "immune checkpoint" and "anti-immune checkpoint therapy" are described above.
In still another embodiment, immunomodulatory drugs, such as immunocytostatic drugs, glucocorticoids, cytostatics, immunophilins and modulators thereof (e.g., rapamycin, a calcineurin inhibitor, tacrolimus, ciclosporin (cyclosporin), pimecrolimus, abetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate (doca) aldosterone, a non-glucocotticoid steroid, a pyrimidine synthesis inhibitor, leflunomide, teriflunomi de, a folic acid analog, methotrexate, anti-thymocyte globulin, anti-lymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, an opioid, an IMPDH inhibitor, mycophenolic acid, myriocin, fingolimod, an NF-xl3 inhibitor, raloxifene, drotrecogin alfa, denosumab, an NF-xB signaling cascade inhibitor, disulfiram, olmesartan, dithiocarbamate, a proteasome inhibitor, bortezomib, MG132, Prol, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomi de, flavopiridol, non-steroidal anti-inflammatory drugs (NSAIDs), arsenic trioxide, dehydroxymethylepoxyquinomycin (DHMEQ), 13C(indole-3-carbinol)/DIM(di-indolmethane) (13C/D1M), Bay 11-7082, luteolin, cell permeable peptide SN-50, IKBa.-super repressor overexpression, NFKB decoy oligodeoxynucleotide (ODN), or a derivative or analog of any thereo, are used. In yet another embodiment, immunomodulatory antibodies or protein are used. For example, antibodies that bind to CD40, Toll-like receptor (TLR), 0X40, GITR, CD27, or to 4-1BB, T-cell bispecific antibodies, an anti-LL-2 receptor antibody, an anti-CD3 antibody, OKT3 (muromonab), otelixizumab, teplizumab, visilizumab, an anti-CD4 antibody, clenoliximab, keliximab, zanolimumab, an anti-CD11 a antibody, efalizumab, an anti-CD18 antibody, erlizumab, rovelizumab, an anti-antibody, afutuzumab, ocrelizumab, ofatumumab, pascolizumab, rituximab, an anti-CD23 antibody, lumiliximab, an anti-CD40 antibody, teneliximab, toralizumab, an anti-CD4OL
antibody, ruplizumab, an anti-CD62L antibody, aselizumab, an anti-CD80 antibody, galiximab, an anti-CD147 antibody, gavilimomab, a B-Lymphocyte stimulator (BLyS) inhibiting antibody, belimumab, an CTLA4-Ig fusion protein, abatacept, belatacept, an anti-CTLA4 antibody, ipilimumab, tremelimumab, an anti-eotaxin 1 antibody, bertilimumab, an anti-a4-integrin antibody, natalizumab, an anti-IL-6R antibody, tocilizumab, an anti-LFA-1 antibody, odulimomab, an anti-CD25 antibody, basiliximab, daclizumab, inolimomab, an anti-CD5 antibody, zolimomab, an anti-CD2 antibody, siplizumab, nerelimomab, faralimomab, atlizumab, atorolimumab, cedelizumab, dorlimomab aritox, dorlixizumab, fontolizumab, gantenerumab, gomiliximab, lebrilizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, talizumab, telimomab aritox, vapaliximab, vepalimomab, aflibercept, alefacept, rilonacept, an IL-1 receptor antagonist, anakinra, an anti-IL-5 antibody, mepolizumab, an IgE inhibitor, omalizumab, talizumab, an 11,12 inhibitor, an IL23 inhibitor, ustekinumab, and the like.
Nutritional supplements that enhance immune responses, such as vitamin A, vitamin E, vitamin C, and the like, are well known in the art (see, for example, U.S.
Pat. Nos.
4,981,844 and 5,230,902 and PCT Pub!. No. WO 2004/004483) can be used in the methods described herein.
Similarly, compositions and therapies other than immunotherapy or in combination thereof can be used with in combination with the compositions of the present invention to stimulate an immune response to thereby treat a condition that would benefit therefrom.
For example, chemotherapy, radiation, epigenetic modifiers (e.g., histone deacetylase (HDAC) modifiers, methylation modifiers, phosphorylation modifiers, and the like), targeted therapy, and the like are well known in the art.
In one embodiment, chemotherapy is used. Chemotherapy includes the administration of a chemotherapeutic composition. Such a chemotherapeutic composition may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolities, anti-mitotic compositions, alkylating compositions, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof.
Exemplary compounds include, but are not limited to, alkylating compositions:
cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA
topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; anti-folates:
methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs: mercaptopurine and thioguanine; DNA
antimetabolites: 2'-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole;
and antimitotic compositions: halichondrin, colchicine, and rhizoxin.
Compositions comprising one or more chemotherapeutic compositions (e.g., FLAG, CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP

comprises cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiments, PARP (e.g., PARP-1 and/or PARP-2) inhibitors are used and such inhibitors are well known in the art (e.g., Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research Laboratories, Inc.); INO-1001 (Inotek Pharmaceuticals Inc.); PJ34 (Soriano et al., 2001;
Pacher et al, 2002b); 3-aminobenzamide (Trevigen); 4-amino-1,8-naphthalimide;
(Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re.
36,397); and NU1025 (Bowman et al.). The mechanism of action is generally related to the ability of PARP inhibitors to bind PARP and decrease its activity. PARP catalyzes the conversion of .beta.-nicotinamide adenine dinucleotide (NAD+) into nicotinamide and poly-ADP-ribose (PAR). Both poly (ADP-ribose) and PARP have been linked to regulation of transcription, cell proliferation, genomic stability, and carcinogenesis (Bouchard V. J.
et.al. Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 477, Number 1, 2 Jun. 2001, pp. 97-110(14)). Poly(ADP-ribose) polymerase 1 (PARP1) is a key molecule in the repair of DNA single-strand breaks (SSBs) (de Murcia J. et al.
1997. Proc Natl Acad Sci USA 94:7303-7307; Schreiber V, Dantzer F, Ame J C, de Murcia G
(2006) Nat Rev Mol Cell Biol 7:517-528; Wang Z Q, et al. (1997) Genes Dev 11:2347-2358).
Knockout of SSB repair by inhibition of PARP1 function induces DNA double-strand breaks (DSBs) that can trigger synthetic lethality in cancer cells with defective homology-directed DSB repair (Bryant HE, et al. (2005) Nature 434:913-917; Farmer H, et al. (2005) Nature 434:917-921). The foregoing examples of chemotherapeutic compositions are illustrative, and are not intended to be limiting. Additional examples of chemotherapeutic and other anti-cancer compositions are described in US Pat. Publs.
2013/0239239 and 2009/0053224.
In still another embodiment, the term "targeted therapy" refers to administration of compositions that selectively interact with a chosen biomolecule to thereby treat cancer.
For example, bevacizumab (AvastinO) is a humanized monoclonal antibody that targets vascular endothelial growth factor (see, for example, U.S. Pat. Publ.
2013/0121999, WO
2013/083499, and Presta etal. (1997) Cancer Res. 57:4593-4599) to inhibit angiogenesis accompanying tumor growth. In some cases, targeted therapy can be a form of immunotherapy depending on whether the target regulates immunomodulatory function.

The term "untargeted therapy" referes to administration of compositions that do not selectively interact with a chosen biomolecule yet treat cancer.
Representative examples of untargeted therapies include, without limitation, chemotherapy, gene therapy, and radiation therapy.
Regarding irradiation, a sublethal dose of irradiation is generally within the range of 1 to 7.5 Gy whole body irradiation, a lethal dose is generally within the range of 7.5 to 9.5 Gy whole body irradiation, and a supralethal dose is within the range of 9.5 to 16.5 Gy whole body irradiation.
Depending on the purpose and application, the dose of irradiation may be administered as a single dose or as a fractionated dose. Similarly, administering one or more doses of irradiation can be accomplished essentially exclusively to the body part or to a portion thereof, so as to induce myeloreduction or myeloablation essentially exclusively in the body part or the portion thereof. As is widely recognized in the art, a subject can tolerate as sublethal conditioning ultra-high levels of selective irradiation to a body part such as a limb, which levels constituting lethal or supralethal conditioning when used for whole body irradiation (see, for example, Breitz (2002) Cancer Biother Radiopharm.
17:119; Limit (1997)J. Nucl. Med. 38:1374; and Dritschilo and Sherman (1981) Environ.
Health Perspect 39:59). Such selective irradiation of the body part, or portion thereof, can be advantageously used to target particular blood compartments, such as specific lymph nodes, in treating hematopoietic cancers.
The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16:
Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds., J B. Lippencott Company, Philadelphia. The radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. The radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. Also encompassed is the use of photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and DMHA.
In another embodiment, hormone therapy is used. Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH
antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A
derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs;
antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).
IV. Administration of Compositions of Matter - cyclic di-nucleotide svnthetase enzyme containing Vectors, Pharmaceutical Compositions, Vaccine, Adjuvants The compositions of the invention (e.g., the recombinant vectors (e.g., any gene therapy vectors), containing at least one nucleic acid encoding a cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or a portion or ortholog thereof, and pharmaceutical compositions, vaccines, and adjuvants comprising same) are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo, to either enhance immune cell mediated immune responses. By "biologically compatible form suitable for administration in vivo" is meant a form of the compositions described herein to be administered in which any toxic effects are outweighed by the therapeutic effects of the compositions. The term "subject" is intended to include living organisms in which an immune response can be elicited, e.g., mammals.
Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
Administration of a compositions as described herein can be in any pharmacological form including a therapeutically active amount of an composition alone or in combination with a pharmaceutically acceptable carrier.
Administration of a therapeutically active amount of the therapeutic composition of the present invention is defined as an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, a therapeutically active amount of a vaccine may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of peptide to elicit a desired response in the individual. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.
The compositions of the present invention described herein can be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration.
Depending on the route of administration, the active compound can be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. For example, for administration of compositions, by other than parenteral administration, it may be desirable to coat the composition with, or co-administer the composition with, a material to prevent its inactivation.
An composition can be administered to an individual in an appropriate carrier, diluent or adjuvant, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Additional adjuvants may to combine with the compositions of the present invention include resorcinols, non-ionic surfactants such as polyoxyethylene ()ley] ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Sterna et al. (1984)J.
Neuroimmunol. 7:27).
The composition may also be administered parenterally or intraperitoneally.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
Pharmaceutical compositions of compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the composition will preferably be sterile and must be fluid to the extent that easy syringeability exists. It will preferably be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compositions, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic compositions, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought about by including in the composition an composition which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating a composition of the present invention (e.g., vector (e.g., any gene therapy vector comprising at least one cyclic di-nucleotide synthetase enzyme, such as AdVCA0956 or AdVCA0848)) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by =
incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the composition plus any additional desired ingredient from a previously sterile-filtered solution thereof.
When the composition is suitably protected, as described above, the protein can be orally administered, for example, with an inert diluent or an assimilable edible carrier. As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal compositions, isotonic and absorption delaying compositions, and the like. The use of such media and compositions for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or composition is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form", as used herein, refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present invention are dictated by, and directly dependent on, (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
In one embodiment, a composition of the present invention is a vector (e.g., any gene therapy vector comprising at least one cyclic di-nucleotide synthetase enzyme, such as AdVCA0956 or AdVCA0848). As defined herein, a therapeutically effective amount of the adenovirus (i.e., an effective dosage) ranges from about 1x104 to 1x1012 infectious particles/kg. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a vector (e.g., any gene therapy vector comprising at least one cyclic di-nucleotide synthetase enzyme, such as AdVCA0956 or AdVCA0848) can include a single treatment or, preferably, can include a series of treatments. In some embodiments, a subject is treated with a vector (e.g., any gene therapy vector comprising at least one cyclic di-nucleotide synthetase enzyme, such as AdVCA0956 or AdVCA0848) in the range of between about lx iO4 to lx1012 infectious particles/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of vector (e.g., any gene therapy vector comprising at least one cyclic di-nucleotide synthetase enzyme, such as AdVCA0956 or AdVCA0848) used for treatment may increase or decrease over the course of a particular treatment.
Changes in dosage may result from the results of diagnostic assays. In addition, a vector (e.g., any gene therapy vector comprising at least one cyclic di-nucleotide synthetase enzyme, such as AdVCA0956 or AdVCA0848) of the present invention can also be administered in combination therapy with, e.g., chemotherapeutic compositions, hormones, antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy, and/or radiotherapy.
A vector (e.g., any gene therapy vector comprising at least one cyclic di-nucleotide synthetase enzyme, such as AdVCA0956 or AdVCA0848) of the present invention can also be administered in conjunction with other forms of conventional therapy, either consecutively with, pre- or post-conventional therapy. For example, the vector (e.g., any gene therapy vector comprising at least one cyclic di-nucleotide synthetase enzyme, such as AdVCA0956 or AdVCA0848) can be administered with a therapeutically effective dose of chemotherapeutic composition. In another embodiment, the vector (e.g., any gene therapy vector comprising at least one cyclic di-nucleotide synthetase enzyme, such as AdVCA0956 or AdVCA0848) can be administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic composition. The Physicians' Desk Reference (PDR) discloses dosages of chemotherapeutic compositions that have been used in the treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular immune disorder being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.
In addition, the compositions of the present invention described herein can be administered using nanoparticle-based composition and delivery methods well known to the skilled artisan. For example, nanoparticle-based delivery for improved nucleic acid therapeutics are well known in the art (Expert Opinion on Biological Therapy 7:1811-1822).
V. Kits The present invention also encompasses kits for treating disorders that would benefit from upregulated immunot responses, such as pathogenic infections and cancers, using the compositions of the invention (e.g., the recombinant vectors (e.g., adeonovirial vectors), containing a nucleic acid encoding a cyclic di-nucleotide synthetase enzyme (e.g., DGCs, DACs, Hypr-GGDEFs, DncV, DisA, cGAS, any sequences that encode GGDEF
domains belonging to the COG2199 protein domain family) listed herein, the Figures, and the Examples, or any subset thereof, or a portion or ortholog thereof, and pharmaceutical compositions, vaccines, and adjuvants comprising same). For example, the kit can comprise the recombinant vectors (e.g., any gene therapy vector comprising at least one cyclic di-nucleotide synthetase enzyme, such as AdVCA0956 or AdVCA0848, extracellular antigen, or Ad containing Ag) in hydrophilized, dried, or liquid form that is packaged in a suitable container. The kit can further comprise instructions for using such compositions to treat pathogenic infections and/or cancers in a patient in need thereof. The kit may also contain other components, such as administration tools like packaged in a separate container.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference.
Exemplification This invention is further illustrated by the following examples, which should not be construed as limiting.
Example 1: Materials and Methods for Examples 2-5 All of the DNA manipulation and plasmid construction was performed as previously described (Sambrook J etal. (2001) Molecular Cloning - A Laboratory Manual, 3rd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The VCA0956 gene was amplified from Vibrio cholerae El tor strain C6706 using the DNA polymerase Phusion (New England Biolabs) and the oligonucleotides 5'-ATAGGTACCCCACCGTGATGACAACTGAAGATTTCA-3' and 5'-ATACTCGAGTTAGAGCGGCATGACTCGAT-3' (IDT). This product was then inserted into the plasmid pShuttle-CMV (Seregin SS et al. (2010) Hum. Gene Ther.
22:1083-1094) by digesting with Kpnl and XhoI (Fermentas), and then ligated with a T4 DNA
ligase (Invitrogen). Eseherichia colt strain DH1OB (Invitrogen) was used for harboring plasmid DNA, and sequence fidelity was confirmed by sequencing (Genewiz). The active site mutant allele was generated using the QuickChange Lightning site-directed mutagenesis kit (Agilent) with the primer 5'-TGACAGCTTATCGTTATGCCGCTGAAGAGTTTGCACTGAT-3'.
A first-generation, human Ad type 5 (Ad5) replication deficient vector (deleted for the El and E3 genes) was used in this study (Seregin SS etal. (2009) Gene Ther. 16:1245-1259). Recombination, viral propagation of the Ad5 vectors, and subsequent virus characterization was performed as previously described (Seregin SS etal.
(2009) Gene Hier. 16:1245-1259; Seregin SS etal. (2010) Blood 116:1669-1677). Viral particle number was determined by optical density measurement at 260 nm and validated as previously described (Amalfitano A etal. (1998)J. Virol. 72:926-933).Construction of the Ad5-Null and Ad5-TA is described elsewhere (Morgan J et al. (2002) Construction of First-Generation Adenoviral Vectors, p. 389-414, Gene Therapy Protocols, vol. 69.
Springer New York; Seregin SS et al. (2012) Vaccine 30:1492-1501). All virus constructs were confirmed to be replication-competent adenovirus (RCA) negative using RCA PCR
and direct sequencing methods (Seregin SS et al. (2009) Gene Ther. 16:1245-1259) and the bacterial endotoxin content was found to be < 0.15 EU per mL (Seregin SS et al. (2009) Gene Ther. 16:1245-1259). All procedures with recombinant adenovirus constructs were performed under BSL-2 conditions, All transfections of plasmid DNA into HeLa cells was performed with the TransIT-HeLaMONSTER transfection kit (Mirus) in 6-well plates with 2.5 vg plasmid DNA.
For HeLa cell infections with adenovirus vectors, cells were infected with 2.0*
109 viral particles (M.O.I. of 500). Cell cultures were checked for confluence and morphology before and after transfection and infection using microscopy. After 24 hours of growth at 37 C in 5% CO2, the cells were dissociated using 300 i.LL 0.25% trypsin, and then cells were resuspended in 4 mL PBS and then pelleted by centrifugation at 1600 RPM at 4 C.
Afterwards the cells were resuspended in 100 I, extraction buffer (40%
acetonitrile, 40%
methanol, and 0.1 N formic acid). The cell lysate was incubated at -20 C for 30 minutes, and then centrifuged at max speed for 10 minutes. The extraction buffer was removed from the pelleted debris and stored at -80 C until analysis.
Immediately prior to analysis, the extraction buffer was evaporated using a vacuum manifold, and the samples were rehydrated in 1004 water. C-di-GMP was quantified using an Acquity Ultra Performance liquid chromatography system (Waters) coupled with a Quattro Premier XE mass spectrometer (Waters) as previously described (Massie JP et al.
(2012) Proc. Natl. Acad. Sci. U. S. A. 109:12746-12751), The concentration of c-di-GMP
was determined by generating an 8-point standard curve (1:2 dilutions) of chemically synthesized c-di-GMP (Biolog) ranging from 1.9 to 250 nM. The intracellular concentration was estimated by dividing the total molar amount of c-di-GMP extracted by the estimated total intracellular volume of HeLa cells extracted using cell counts and size measurements determined using a Countess Automated cell counter (Life Technologies). The transfection efficiency was determined to be 18.2%, which was obtained by transfecting HeLa cells with plasmid containing GFP under CMV promoter control and measuring the percent of GFP
positive cells using flow cytometry. The infection efficiency of HeLa cells was determined to be 82.2%, which was determined by infecting HeLa cells with Ad5-gfl, (Seregin SS et al.

(2010) Blood 116:1669-1677) and quantifying the percent of GFP positive cells using flow cytometry.
Adult BALB/c WT male mice (6-8 weeks old) were used for all animal experiments (Jackson Laboratory). For c-di-GMP quantification and innate studies, mice were anesthetized using isofluorane, and 2x1011 adenovirus viral particles (vp) per mouse (200 I, total volume, suspended in lx sterile PBS) were administered intravenously (IV) via retro-orbital injection. After administration, mice were monitored every 6 hours by lab personnel for mortality and other health parameters in accordance with Michigan State University EHS and IACUC. After 24 hours the mice were sacrificed, and the spleen and the left lobe of the liver were isolated from each animal. Each tissue was placed in 500 L
PBS, and then the tissue suspension was homogenized using an Omni Tissue Homogenizer (Omni International). 300 I. of homogenate was added to an equal volume of equilibrated Phenol Solution (Sigma). The homogenate-phenol solution was vortexed and centrifuged at 15,000 rpm for 10 minutes. The aqueous phase was removed and added to 500 I, chloroform. The mixture was vortexed and then centrifuged at 15,000 rpm for 10 minutes.
The aqueous phase was then removed and stored at -80 C until analysis.
Quantitative PCR was used to determine adenovirus abundance from DNA
extracted from liver tissue as previously described (Seregin SS et al. (2009) Mot Ther.
17:685-696). Ad5 genome copy numbers were quantified using an ABI 7900HT Fast Real-Time PCR system and the SYBR Green PCR Mastermix (Applied Biosystems) in a 15 L
reaction using a primer set for the Ad5 Hexon gene that has been previously described (Appledorn DM etal. (2008) Gene Ther. 15:885-901). All PCRs were subjected to the following procedure: 95.0 C for 10 minutes, followed by 40 cycles of 95.0 C
for 15 seconds and 60.0 C for 1 minute. Standard curves to determine the number of viral genomes per liver cell were run in duplicate and consisted of 6 half-log dilutions using DNA extracted from purified Ad5 virus (Seregin SS et al. (2009) Gene Ther.
16:1245-1259). As an internal control, liver DNA was quantified using primers spanning the GAPDH gene (Seregin SS etal. (2009) Mot Ther. 17:685-696) and standard curves were generated from total genomic DNA. Melting curve analysis was performed to confirm the quality and specificity of the PCR (data not shown).
To determine relative abundance of specific liver-derived RNA transcript, reverse transcription was performed on RNA derived from the liver tissue using SuperScript III
(Invitrogen) and random hexamers (Applied Biosystems) as per the manufacturer's instruction. RT reactions were diluted to a total volume of 60 tL, and 2 pt from each sample was used as template for subsequent PCR. Quantitative PCR was subsequently performed as described above using an AI31 7900HT Fast Real-Time PCR system and SYBR Green PCR Mastermix (Applied Biosystems) using primer sets that have been previously described (Seregin SS etal. (2009) Gene Ther. 16:1245-1259). The comparative Ct method was used to determine relative gene expression using GAPDH to standardize expression levels across all samples. Relative expression changes were calculated by comparing experimental levels of liver transcript to levels of liver transcript derived from mock-treated animals.
IFN-I3 was quantified using the Verikine Mouse IFN Beta ELISA kit (PBL Assay Science) as per manufacturer's instruction. Cytokine and chemokine concentrations were quantified from plasma samples using a Bio-Plex multiplex bead array system (Bio-Rad).
At 6 and 24 hours, blood samples were taken from mice using heparinized capillary tubes and EDTA-coated microvettes (Sarstedt). The samples were centrifuged at 3,400 rpm for 10 minutes to isolate plasma. Samples were assayed for 12 independent cytolcines and chemokines (IL-la, IL-4, IL-6, IL12-p40, IFN-y, G-CSF, Eotaxin, KC, MCP-1, MIP-1a, 1VIIP-113, and RANTES) as per the manufacturer's instructions (Bio-Rad) via Luminex 100 technology (Luminex).
For adaptive immunity studies, mice were administered adenovirus ranging from 1x106 to 5x109vp per mouse suspended in 25 jiL PBS via IM injection into the tibialis anterior of the right hindlimb. To measure antigen specific recall responses, mice were sacrificed and the spleen was harvested after 14 days. Splenocytes were isolated and ex vivo stimulated with immunogenic peptides from C. difficik TA library as previously described (Seregin SS etal. (2012) Vaccine 30:1492-1501). ELISpot analysis was performed as previously described (Seregin SS etal. (2012) Vaccine 30:1492-1501) using 96-well multiscreen high-protein binding Immobilon-P membrane plates (Millipore) and the Ready-Set Go IFN-y mouse ELISpot kit (eBioscience). Spots were photographed and counted using an automated ELISpot reader system (Cellular Technology). To determine TA-specific IgG titers, ELISA based tittering was used on plasma samples taken from the mice 14 d.p.i as previously described (Seregin SS etal. (2012) Vaccine 30:1492-1501).
All animal procedures were reviewed and approved by the Michigan State University EHS and IACUC. Care for the mice was provided in accordance with PHS and AAALAC standards. Plasma and tissue samples were collected and handled in accordance with the Michigan State University Institutional Animal Care and Use Committee.
Example 2: Generating an adenovirus harboring a V. cholerae DGC
Cdi-GMP is an exciting new adjuvant that stimulates the innate immune system (Chen WX et al. (2010) Vaccine 28:3080-3085). These studies most frequently used chemically synthesized c-di-GMP. Because c-di-GMP is synthesized from GTP and GTP is abundant in the cytoplasm of eukaryotic organisms, it was postulated that a DGC expressed under the control of a strong eukaryotic promoter/enhancer element would lead to c-di-GMP synthesis within the eukaryotic cell and subsequent enhancement of downstream innate immune responses. This approach would offer a novel, alternative method to administer c-di-GMP as a vaccine adjuvant as opposed to direct delivery of the synthesized molecule. To identify a DGC that would produce c-di-GMP in the cytoplasm of a eukaryotic cell, DGCs from V cholerae was examined, as V cholerae is a well-studied model system for c-di-GMP signaling and many V. cholerae DGCs have been shown to synthesize c-di-GMP in high concentrations (Massie JP etal. (2012) Proc. Natl.
Acad. Sc!.
U. S. A. 109:12746-12751) The DGC VCA0956 was selected due to the fact that it had no predicted N-terminal regulatory or trans-membrane domains. Furthermore, VCA0956 has a canonical G-GDEF domain and active site motif, and ectopic expression of VCA0956 has been shown to increase biofilm formation in both V. cholerae and Vibrio vulnificus (Massie JP et al. (2012) Proc. Natl. Acad. Sc!. U. S. A. 109:12746-12751; Nakhamchik A
et al.
(2008) App!. Environ. Microbia 74:4199-4209), repress motility in V. cholerae (Hunter JL
et al. (2014) BMC Microbia 14:22), and increase intracellular c-d-GMP in V.
cholerae and Shewanella oneidensis (Koestler BJ etal. (2013) App!. Environ. Microbia 79:5233-5241; Tamayo R et al. (2008) Infect. lmmun. 76:1617-1627; Thormann KM et al.
(2006)1 Bacteria 188:2681-2691).
To determine if VCA0956 is able to synthesize c-di-GNfP in a eukaryotic cytoplasm, a plasmid containing VCA0956 under the control of the constitutive CMV
promoter/enhancer in the plasmid pShuttleCMV was constructed. A second vector containing the same VCA0956 allele with a mutation in the active site of the GGDEF
domain (GGEEF -> AAEEF) was also constructed. These plasmids were transfected into HeLa cells, and c-di-GMP levels were measured in cell lysates after 24 hours using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). It was found that eukaryotic cells transfected with the VCA0956 allele produced detectable levels of c-di-GMP (Figure 1A). In contrast, no detectable c-di-GMP was observed in both cells transfected with the active site mutant allele or a mock treatment controls (Figure 1A). The estimated intracellular c-di-GMP concentrations of HeLa cells grown in 6-well dishes expressing VCA0956 are greater than the Kd range of the c-di-GMP binding protein STING
(2.5 - 4.9 M) (Burdette DL et al. (2011) Nature 478:515-518; Yin Q et al.
(2012) Mol.
Cell 46:735-745).Cell cultures were checked by microscopy and no discernible morphological differences was observed between expression of VCA0956 and the controls.
Furthermore, trypan blue staining indicated that treatment with VCA0956 did not appear to impact overall cell viability. Additionally, HeLa cells grown in t75 flasks transfected with the VCA0956 plasmid and measured 48 hours later had less intracellular c-di-GMP, suggesting that c-di-GMP synthesis is transient (Figure 1B). It was speculated that c-di-GMP could be degraded in eukaryotic cells by nonspecific phosphodiesterase enzymes.
Less c-di-GMP production in these experiments was observed which may be a function of decreased transfection efficiency in the flasks. Nevertheless, these results indicate that VCA0956 is capable of transiently synthesizing c-di-GMP in the cytoplasm of eukaryotic cells grown in vitro.
The pShuttleCMV-VCA0956 plasmid and its mutant allele counterpart were then used to construct and purify to high concentration the respective recombinant Ad5-based vectors. To confirm that the VCA0956 Ad5 construct, herein referred to as Ad5-VCA0956, was able to produce c-di-GMP in a eukaryotic cytoplasm, HeLa cells (500 multiplicity of infection, M.O.I.) were infected with the Ad5-VCA0956 and Ad5-VCA0956 mutant allele (Ad5-VCA0956*) adenovirus vectors and measured c-di-GMP using LC-MS/MS after hours. The Ad5-Null vector, an adenovirus construct carrying no transgene, was also included as a negative control. It was found that cells infected with the Ad5-produced high concentrations of c-di-GMP comparable to transfection of the pShuttleCMV-VCA0956 plasmid, whereas cells infected with the Ad5-VCA0956* or the Ad5-Null produced no detectable c-di-GMP (Figure 2). Importantly, similar to VCA0956 plasmid transfections, infection with Ad5-VCA0956 had no noticeable impact on cell morphology or viability. These results demonstrate that an adenovirus vector can be used to deliver VCA0956 into HeLa cells to synthesize c-di-GMP.
Example 3: Synthesis of c-di-GMP in vivo As the Ad5-VCA0956 vector is capable of producing c-di-GMP in HeLa cells in vitro, it was next determined if this vector produces c-di-GMP in vivo in a murine model system. BALB/c mice (n = 3) were IV injected with the Ad5-Null, Ad5-VCA0956, or the Ad5-VCA0956* vectors and quantitative PCR was utilized to measure adenovirus genomes in the spleen and liver of injected mice at 24 hours post injection (h.p.i.).
Using quantitative RT-PCR comparable Ad5 genome counts were observed for each treatment in both the liver and spleen (Figure 3A). Consistent with previous reports that the predominant tropism of adenovirus is in the liver (Appledorn DM etal. (2008) Gene Ther. 15:885-901;
Everett RS
et al. (2003) Hum. Gene Ther. 14:1715-1726; Nakamura T et al. (2003)J. Virol.
77:2512-2521) there were significantly more Ad5 genomes in the liver cells than in the spleen cells.
C-di-GMP in both the liver and spleen using LC-MS/MS was then measured, and found that the Ad5-VCA0956 vector produced detectable c-di-GMP in both tissues, whereas the Ad5-Null and Ad5-VCA0956* vectors produced no detectable c-di-GMP (Figure 3B).
The concentration of c-di-GMP was consistent with the abundance of Ad5-VCA0956 genomes per cell, as the amount of c-di-GMP was significantly higher in the liver tissue than the spleen. These data indicate that the Ad5-VCA0956 vector is capable of initiating c-di-GMP
synthesis in a mouse.
Example 4: e-di-GMP synthesized in vivo stimulates innate immunity in a mouse model It has been previously shown that adenovirus vectors stimulate several pro-inflammatory innate immune response genes (Hartman ZC etal. (2008) Virus Res.
132:1-14; Seregin SS et al (2009) Gene Ther. 16:1245-1259; Seregin SS etal. (2009) Mol. Ther.
17:685-696). To examine if the Ad5-VCA0956 alters the profile of innate immune gene expression compared to the Ad5 vector alone, Balb/c mice (n = 3) were IV
injected with Ad5-Null, Ad5-VCA0956, and Ad5-VCA0956* and qRT-PCR was utilized to quantify the expression levels of several liver gene transcripts at 24 hours post infection (h.p.i.).
Infection with Ad5-VCA0956 had no observable effect on the health of the mice.
It was found that the Ad5-Null treatment was able to stimulate 6 of the 12 markers examined (>2-fold; ADAR, MCP-1, TLR2, LP10, Oasla, RIG1) (Figure 4). These results are consistent with previous studies demonstrating that the adenovirus vector alone is capable of altering gene expression in the liver (Seregin SS etal. (2010) Hum. Gene Ther. 22:1083-1094;
Seregin SS et al. (2009) Gene Ther. 16:1245-1259). The expression of four genes was significantly (p <0.05) higher in the Ad5-VCA0956 treatment compared to the Ad5-VCA0956* treatment (Figure 4A); these include the IFN-responsive gene ADAR, the monocyte and basophil chemotractant protein-1 MCP-1, the toll-like receptor (TLR) signaling pathway gene MyD88, and the pattern recognition receptor TLR2. It is worth noting that c-di-GMP sensing in the cytoplasm is thought to be independent of TLRs (Karaolis DKR et al. (2007)1 Immunol. 178:2171-2181). Additionally, the expression of three genes was significantly (p < 0.05) repressed in the Ad5-VCA0956 treatment compared to the Ad5-VCA0956* treatment (Figure 4B): the pro-inflammatory interleukin genes IL18 and 1L113, and the interferon transcription factor IRF3.
Interestingly, IRF3 has been shown to interact with STING to initiate a c-di-GMP-mediated host type I
interferon response (McWhiner SM etal. (2009)J Exp. Med. 206:1899-1911; Tanaka Y et al.
(2012) Sci. Signal. 5:ra20; de Almeida LA et al. (2011) PLoS ONE 6:e23135).
In the cytoplasm, c-di-GMP interacts with STING to initiate a type-I
interferon response and activates IRF3, NF-x13, and the p38/JNK/ERK MAP kinase signaling pathways, resulting in increased production of numerous cytokines and chemokines (McWhirter SM etal. (2009) J. Exp. Med 206:1899-1911). To determine if Ad5-is capable of inducing type-I interferons, the concentration of IFN-I3 in the plasma of mice I.V. treated with Ad5-Null, Ad5-VCA0956, or Ad5-VCA0956* at 6 h.p.i. and 24 h.p.i.
were measured. It was found that at 6 h.p.i., IFN-13 concentrations were significantly higher in mice treated with Ad5-VCA0956 compared to the other controls (Figure 5). At 24 h.p.i., IFN-I3 concentrations were undetectable in the control mice, whereas mice treated with Ad5-VCA0956 demonstrated IFN-I3 concentrations that were detectable, although lower than those at the 6 h.p.i. timepoint. These data indicate that Ad5-VCA0956 is capable of inducing a type-I interferon response in mice.
In addition to IFN-I3, it was further determined if other cytokines and chemokines were induced by Ad5-VCA0956. To this end, the abundance of cytokines and chemokines in the plasma of mice treated with Ad5-VCA0956 using a multiplexed assay system at 6 and 24 h.p.i. were directly quantified. Consistent with prior studies showing that the adenovirus vector stimulates the secretion of pro-inflammatory cytokines and chemokines (27, 28), it was observed that 9 cytokines and chemokines were modestly induced in the Ad5-Null treated mice compared to the naïve mice (IFN-y, MCP-1, G-CSF, MIP-la, IL-6, MIP-113, IL-12p40, KC, RANTES; > 3-fold), and these differences were greatest at the 6-hour time point (Figure 6). It wa found that12 cytokines and chemokines, shown in Figure 6 were significantly increased in the plasma of the Ad5-VCA0956 treated mice compared to the control Ad5-VCA0956* treated mice at one or both of the two timepoints.
Furthermore, for the majority of cytokines and chemokines examined, the largest differences observed were at the 24 hour time point, indicating that the effect of Ad5-VCA0956 is both more potent and longer lasting than that of the adenovirus vector alone. The induction of most of these cytokines and chemokines are consistent with other studies examining the immunostimulatory effects of c-di-GMP (Ebensen T etal. (2007) Vaccine 25:1464-1469;
Ebensen T etal. (2007) Cl/n. Vaccine Immunol. 14:952-958; Karaolis DKR et al.
(2007)J.
Immunol. 178:2171-2181; Karaolis DKR etal. (2007) Infect. Immun. 75:4942-4950;
Yan 11B et al. (2009) Biochem. Biophys. Res. Commun. 387:581-584; Gray PM etal.
(2012) Cell Immunol. 278:113-119). Interestingly, increases in 1L-la, G-CSF, and Eotaxin levels in the Ad5-VCA0956 injected mice were observed, which have not been previously reported to be induced by c-di-GMP. These data together indicate that the Ad5-vector is capable of inducing a robust innate response beyond that of the adenovirus vector alone in a murine model system.
Example 5: Ad5-VCA0956 lowers the effective dose for a T-cell response to a Clostridium difficile antigen The function of an adjuvant is to enhance the efficacy of a paired antigen by increasing the longevity, potency, or reducing the effective dose. Previous data showed that Ad5-VCA0956 strongly upregulates inflammatory responses. To test if the Ad5-construct functions as a vaccine adjuvant, it was determined if Ad5-VCA0956 could enhance the adaptive response to a C. difficile antigen. C. difficile, a Gram-positive spore-forming anaerobic bacteria, is the leading causative composition of nosocomial infections leading to diarrhea] disease in the developed world. C. difficile associated diarrhea (CDAD) represents nearly 1% of all hospital stays in the United States and can lead to septicemia, renal failure, and toxic megacolon (Lucado I et al. (2012. Clostridium dOrkile Infections (CDI) in Hospital Stays, 2009. Agency for Healthcare Research and Quality).
Incidents and mortality of C. difficile infections are rising in the U.S., and the economic burden on the health care system is reported to be in the billions of dollars (Lucado J et al. (2012.
Clostridium difficile Infections (CDI) in Hospital Stays, 2009. Agency for Healthcare Research and Quality; Morris AM et al. (2002) Arch. Surg. 137:1096-1100;
Redelings MD
etal. (2007) Increase in Clostridium difficile¨related mortality rates, United States, 1999-2004. Emerg Infect Dis; Kyne L et al. (2002) Clin. Infect. Dis. 34:346-353;
Dubberke ER et al. (2009) Epidemiol. 30:57-66). Furthermore, to date there are no approved effective vaccine treatments available for CDAD treatment or prevention (Aslam S et al.
(2005) Lancet Inf. Dis. 5:549-557).
An adenovirus vector that expresses the immunogenic portion of the C.
difficile toxin A (Ad5-TA) was previously developed and demonstrated to protect mice from a toxin challenge by generating a humoral and T-cell response specific to toxin A in a murine model system (Seregin SS et al. (2012) Vaccine 30:1492-1501). It was hypothesized that supplementing this vaccine with the Ad5-VCA0956 adjuvant would enhance this humoral and T-cell response due to the strong innate immune stimulatory activity of VCA0956.
Therefore mice were vaccinated by IM injection with varying concentrations of the Ad5-TA vector in combination with the Ad5-VCA0956 vector in equal ratio ranging from lx106 to 5x109 viral particles (vp). After two weeks, TA-specific IgG titers in the plasma of the vaccinated mice were measured. At the lx 107 dose, no significant changes in TA-specific IgG in the plasma of any of the treated mice were observed compared to the mock treatment, indicating that this dose of Ad5-TA and Ad5-VCA0956 is not sufficient to produce a robust IgG response in mice (Figure 7A). In contrast, the 5x109 dose resulted in significantly increased TA-specific IgG in both the Ad5-VCA0956 and Ad5-VCA0956*, however the TA-specific IgG titers in the Ad5-VCA0956* treated animals was modestly higher (2-way ANOVA, p <0.05) than those treated with Ad5-VCA0956 (Figure 7B), suggesting that higher doses of c-di-GMP has a negative impact on humoral immunity.
TA specific T-cell responses in the spleens of the naive and vaccinated animals were also assessed using an IFN-y ELISpot assay, utilizing the 15-mer peptide (VNGSRYYFDTDTAIA) that has been previously shown to elicit the secretion of IFN-y in splenocytes of mice immunized with the Ad5-TA vector (Seregin SS et al .
(2012) Vaccine 30:1492-1501). It was found that co-injection of equal amounts of the Ad5-TA
and the mutant DGC allele vector Ad5-VCA0956* produced no induction of IFN-y secreting cells over that of naïve splenocytes at viral doses of 1x106 and 1x107, but did generate significant IFN-y producing 1-cells at 1x108 and 5x109 (Figure 8, white squares). The number of spot-forming cells (SFCs) in the mice treated with Ad5-TA and Ad5-VCA0956*
at the 5x109 dose was consistent with SFCs of mice vaccinated with Ad5-TA
alone (Seregin SS etal. (2012) Vaccine 30:1492-1501). These data indicate that antigen-specific 1-cells responses in splenocytes plateaus at high levels of Ad5-TA independent of the addition of c-di-GMP. Although co-injection of 1x106 Ad5-TA with Ad5-VCA0956 did not produce increased IFN-y levels, we observed significantly increased (p <0.05) IFN-y producing T-cells at a dose of lx107, as compared to cells derived from the DGC mutant treated control (Figure 8, black squares). However, the number of IFN-y splenocytes did not reach those of the mice injected with higher concentrations of Ad5-TA and Ad5-VCA0956, suggesting only a modest improvement compared to the negative controls. IFN-y producing T-cells at injections of lx108 and 5x109 Ad5-TA and Ad5-VCA0956 were similar to the DGC
mutant control. No c-di-GMP was detected in the liver of mice infected with Ad5-VCA0956 at the 5x109 dose after 14 days, suggesting that even at high doses intramuscular administration of Ad5-VCA0956 does not lead to long-lasting c-di-GMP production at distal sites (data not shown). Thus, it was concluded that although it does not increase a humoral response, c-di-GMP synthesized by Ad5-VCA0956 modestly lowers the effective dose to generate a T-cell response to Ad5-TA in a murine model system.
Discussion With a current demand for novel vaccines that target difficult-to-treat diseases, it is crucial to have adjuvants to pair with these vaccines to optimize efficacy.
Currently, there are a limited number of adjuvants available for clinical use, and there is a need for new adjuvants which can enhance the efficacy of vaccines to improve immunological protection (Coffman RL etal. (2010) Immunity 33:492-503; Reed SG et al. (2009) Trends Immunol.
30:23-32). Numerous studies have implicated c-di-GMP as a promising novel adjuvant.
Indeed, this second messenger molecule has been shown to stimulate a robust type I
interferon response and increase the secretion of numerous cytokines and chemokines to initiate a balanced Thl/Th2 response, as well as stimulate the inflammasome pathway and immune cell activation/recruitment (Sauer JD et al. (2011) Infect. Immun.
79:688-694;
Ebensen T et al. (2007) Vaccine 25:1464-1469; Abdul-Sater AA etal. (2013) EMBO

reports 14:900-906; Ebensen T etal. (2007) Clin. Vaccine Immunol. 14:952-958;
Karaolis DKR etal. (2007) J Immunol. 178:2171-2181; Karaolis DKR et al. (2007) Infect.
Immun.
75:4942-4950; Yan 1-113 et al. (2009) Biochem. Biophys. Res. Commun. 387:581-584; Gray PM etal. (2012) Cell Immunol 278:113-119; Blaauboer SM etal. (2014)1 Immunol.
192:492-502). Described herein is a novel approach in that it utilizes an adenovirus vector to deliver c-di-GMP producing enzyme DNA into cells, thereby synthesizing the adjuvant in vivo. Adenovirus vectors are promising in that they are cost-efficient to produce and can efficiently deliver specific antigens or adjuvants into cells for in vivo production.

It was demonstrated that an adenovirus vector carrying a bacterial DGC is capable of synthesizing c-di-GMP in both human and mouse model systems. Similar to previous studies, it was demonstrated that c-di-GMP synthesized by Ad5-VCA0956 is able to induce a type-I interferon response (Figure 5). Furthermore, synthesis of c-di-GMP by Ad5-VCA0956 increases the secretion of numerous cytokines and chemokines (Ebensen T et al.
(2007) Vaccine 25:1464-1469; Ebensen T et al. (2007) Clin. Vaccine Immunol.
14:952-958;
Karaolis DKR et al. (2007)J. Immunol. 178:2171-2181; Karaolis DKR et al.
(2007) Infect.
Immun. 75:4942-4950; Yan HB etal. (2009) Biochem. Biophys. Res. Commun.
387:581-584; Gray PM et al. (2012) Cell Immunol. 278:113-119; Blaauboer SM et al.
(2014) J.
Immunol. 192:492-502). Importantly, it was demonstrated that Ad5-VCA0956 induces an innate response beyond that of the adenovirus vector alone, which is capable of stimulating the STING system (Lam E et al. (2013)J. Virol. 88:974-981).These cytokines and chemokines induced by Ad5-VCA0956 include signals characteristic of both Thl (e.g. IFN-y, IL-12) and Th2 (e.g. IL-4, IL-6) type responses. Additionally, c-di-GMP
production from Ad5-VCA0956 enhances activation of the innate immune system by activating TLR
signaling (e.g. TLR2, MyD88), It appears however that c-di-GMP synthesized in vivo negatively regulates the expression of inflammasome-dependent pathways in hepatocytes (Figure 4, IL-1)3, IL-18). The significance of this finding is unclear, especially as it has been reported that c-di-GMP activates the NLRP3 inflammasome pathway (Abdul-Sater AA et al. (2013) EMBO reports 14:900-906). Importantly, no signs of poor cell physiology or health were observed in cell cultures and animal models. Furthermore, the data described herein indicated that the c-di-GMP synthesized by the Ad5-VCA0956 vector is transient, and thus should enhance antigen recognition and response while minimizing any potentially unwanted long term effects associated with administration, such as autoimmune activation (53). The mechanism by which c-di-GMP is being eliminated from cell cultures is unknown. It is speculated that native eukaryotic phosphodiesterases are able to hydrolyze the second messenger.
As shown herein, c-di-GMP synthesized in vivo modestly reduces the effective antigen dose of Ad5-TA to produce a T-cell response to a vaccine antigen which targets the toxin of the human pathogen C. difficile. Reducing the dose required to initiate an adaptive immune response is of particular significance as high viral particle doses can lead to global toxicities, endothelial cell activation, and liver damage (Seregin SS et al.
(2009)Mol. Ther.
17:685-696; Everett RS etal. (2003) Hum. Gene Ther. 14:1715-1726; Wolins N et al.

(2003) Br. J. Haematol. 123:903-905; Appledorn DM et al. (2008) i. 15:1606-1617;
Schiedner G et al. (2000) Hum. Gene flier. 11:2105-2116). The data herein suggest that increased c-di-GMP did not enhance the humoral response, however, and modestly decreased antibody production against the C. difficile toxin was observed.
Whether these observations are specific to toxin A from C. difficile or more generally applicable to other antigens is under investigation.
While it was demonstrated that Ad5-VCA0956 is capable of in vivo c-di-GMP
synthesis and has the potential to act as a vaccine adjuvant, further optimization is required to enhance this response. V. cholerae contains 40 predicted DGC alleles within its genome, and it has been shown that ectopic expression of these different DGCs results in different intracellular c-di-GMP concentrations (Massie JP etal. (2012) Proc. Natl.
Acad. Sci. U. S.
A. 109:12746-12751). Hence intracellular expression of other DGCs could produce different amounts of c-di-GMP in eukaryotic cells to optimize the intracellular concentration of c-di-GMP for different applications. Alternatively, other types of second messengers could be used to stimulate innate immunity. One example would be to express a diadenylate cyclase to synthesize the related bacterial second messenger c-di-AMP in vivo.
C-di-AMP has similarly been shown to induce a robust innate immune response through STING mediated recognition (Barker JR etal. (2013) STING-Dependent Recognition of Cyclic di-AMP Mediates Type I Interferon Responses during Chlamydia trachomatis Infection. MBio 4; Woodward JJ etal. (2010) Science 328:1703-1705). Another example is the dinucleotide cyclic guanosine monophosphate¨adenosine monophosphate (cGAMP), a host second messenger produced in response to foreign DNA to activate a STING-dependent type-1 interferon response (Sun L etal. (2012) Science 339:786-791;
Wu Jet al.
(2013) Science 339:826-830; Gao D et al. (2013) Science 341:903-906; Li X-D
etal.
(2013) Science 341:1390-1394). As these second messengers stimulate a STING-mediated innate immune response, they are good alternative candidates for Ad-5 mediated in vivo synthesis. Different promoters could be used in lieu of the CMV promoter to produce localized or temporally controlled c-di-GMP production in the body. Finally, the kinetics of adjuvant production by DGCs and antigen expression could be key factors in stimulating increased adaptive responses.
Other research studies suggest that STING-dependent inflammation inhibits the development of cell-mediated immunity. Archer et. al. recently showed that production of c-di-AMP by the intracellular bacterial pathogen Listeria monocytogenes inhibits cell-.

mediated immunity while inducing inflammatory cytokines in a STING dependent manner (Archer KA et al. (2014) PLoS Pa/hog 10:e1003861). No significant inhibition of either antibody production or IFN-y producing memory T-cells was observed. Whether, these differences are due to the delivery route (L. monocytogenes versus Ad5 transduction), the levels of the signal, or other factors remains to be determined but addressing this question has significant implications for using c-di-GMP or c-di-AMP as a vaccine adjuvant.
C-di-GMP has been shown to enhance protection against other pathogens including S. aureus, K. pneumoniae, and S. pneumoniae (Karaolis DKR etal. (2007)J.
Immunol.
178:2171-2181; Karaolis DKR etal. (2007) Infect. Immun. 75:4942-4950; Yan HB
etal.
(2009) Biochem. Biophys. Res. Commun. 387:581-584; Ogunniyi AD etal. (2008) Vaccine 26:4676-4685), indicating that c-di-GMP has broad antigen-adjuvant synergy.
Although the results of this study imply that that c-di-GMP produced from adenovirus vectors may not enhance vaccines that rely on antibody production, such as those targeting bacterial toxins, the Ad5-VCA0956 stimulated c-di-GMP innate immune response could enhance protection of vaccines that drive cell-mediated immunity such as those targeting viral infections or cancers. Consistent with this idea, c-di-GMP has been shown to exhibit anti-cancer properties in a number of studies (Miyabe H etal. (2014)J. Control. Release 184:20-27;
Chandra D et al. (2014) Cancer Immunology Research. 2(9):901-10; Karaolis DKR
et al.
(2005) Biochem. Biophys. Res. Commun. 329:40-45), which is thought to be mediated through stimulation of a Type I interferon response as observed here. Miyabe et. al. showed that enhancing c-di-GMP entry into cancer cells using liposomes increased its efficacy (Miyabe H etal. (2014) J. Control. Release 184:20-27); adenovirus delivery of DGCs to tumors could function similarly by driving synthesis of c-di-GMP in cancer cells. One advantage of using adenovirus for this purpose over general administration is that modified adenovirus vectors have been constructed to target specific tissue types (Reetz J et al.
(2014) Viruses 6:1540-1563), and c-di-GMP could be directly delivered to tumor cells or other tissue.
Example 6: Materials and Methods for Examples 7-13 1. Vector construction Adenovirus-based vectors used in this study were all replication-deficient.
AdNull and AdGag were constructed as previously described (Aldhamen, YA etal. (2011) J
Immunol 186: 722-732; Seregin, SS et al. (2010) Blood 116: 1669-1677).
AdVCA0848 was constructed similarly to AdVCA0956 as previously described in Examples 1-5.
Briefly, the V. cholerae gene VCA0848 gene (GeneBank sequence: CP007635.1) was sub-cloned into pShuttle-CMV as previously described (Appledorn, DM et al. (2010) PLoS One 5:
e9579).
Primers used for AdVCA0848 construction were: forward: 5'-ATAGGTACCCCACCATGAATGACAAAGTGCT-3' and reverse: 5'-ATACTCGAGTTAGAAAAGTTCAACGTCATCAGAA-3'. The mutant version of AdVCA0848, AdVCA0848m"t, carrying the following amino acid changes: GGEEF >
AAEEF in the GGDEF domain of VCA0848 allele was mutated using the QuikChange Lightning site-directed mutagenesis kit (Agilent) with the primer 5'-GTCTTCTCAACTATTTCGCTTTGCTGCTGAAGAGITCGTGATTATITTTT-3' .
AdToxB was constructed as previously described (Seregin, SS etal. (2012) Vaccine 30:
1492-1501). Briefly, a synthetic gene was designed based on the Clostridium Officile toxin B sequence data from previous studies (Barroso, LA eta!. (1990) Nucleic Acids Res 18:
4004; Kink, JA etal. (1998) Infect Immun 66: 2018-2025) and ordered from GENEART
(Regensburg, Germany). The synthetic gene representing the C-terminal portion of Toxin B, including 617 amino acids (residues 1750-2366), was sub-cloned into pShuttle-CMV as previously described (Appledorn, DM et al. (2010) PLoS One 5: e9579). Primers used for AdToxB construction: forward: 5'-GCTACTACGAGGACGGCCTG-3' and reverse: 5'-CTCATCGATGATCAGCTTGCC-3'. The C-terminal region of the new synthetic gene did not contain the enzymatic domain, and recombination and viral propagation were carried out as described above in Examples 1-5 (Appledorn, DM et al. (2010) PLoS One 5: e9579;
Aldhamen, YA et al. (2012)J Immuno1189: 1349-1359). Constructs were confirmed to be replication-competent adenovirus (RCA) negative using RCA PCR and direct sequencing methods as previously described (Seregin, SS et al. (2010) Blood 116: 1669-1677; Seregin, SS et al. (2009) Mol Ther 17: 685-696). All procedures with recombinant adenovirus constructs were performed under BSL-2 conditions.
2. Animal Procedures The Michigan State University Institutional Animal Care and Use Committee (IACUC) approved the animal procedures conducted in this study. Care was provided to mice in this study in accordance with PHS and AAALAC standards. Mice were purchased from Taconic Biosciences, (Germantown, NY).
To determine the amount of c-di-GMP produced by the AdVCA0848 vector, male 6-8 weeks old Balb/c mice, were intravenously (i.v.) injected (retro-orbitally) with AdNull (n=3), AdVCA0956 (n=4), or AdVCA0848 (n=4) in 200 I of a phosphate-buffered saline solution (PBS, pH 7. 4) containing 2 x1011 viral particles (vps)/mouse; or not injected (naives) (n=3) as previously described (30). The same viral dose was also used for additional experiments in which mice were injected with AdVCA0848, AdVCA0848'1, or not injected (naives). At 24 hours post-injection (hpi), mice were sacrificed and liver samples were collected, immediately snap frozen, and used later for c-di-GMP
quantification as described below.
For innate immunity studies, 6-10 weeks old male C57BL/6 mice (n=4) were iv.
injected (retro-orbitally) with AdNull or AdVCA0848 in 100 1 of a phosphate-buffered saline solution (PBS, pH 7. 4) containing 1x101 vps/mouse or not injected (Naive). The same viral dose was also used for additional experiments in which mice were injected with AdVCA0848, AdVCA0848und, or not injected (naives). At 6 hpi, mice were sacrificed.
Blood samples were collected and used for ELISA analysis and splenocytes were harvested, counted and used for immune cell surface staining. Liver samples were immediately stored at -80 C for c-di-GMP quantification.
To determine the effect of AdVCA0848 on adaptive immune responses against OVA, male 8-10 weeks old C57BL/6 mice (n=4) were co-injected with AdVCA0848 or AdNull in 30 I of a phosphate-buffered saline solution (PBS, pH 7. 4) containing l x1010 vps/mouse via i.m. injection and 100 g/mouse OVA via intraperitoneal (i.p.) injection, with an additional group of mice which were not injected (naives). At 6 days post-injection (dpi), retro-orbital bleeding was used to collect blood samples for ELISA
analysis. At 14 dpi, mice were sacrificed, peripheral blood samples collected and spleen was harvested in 2% FBS RPMI media.
To determine the effect of AdVCA0848 on the adaptive immune response against the HIV-1-derived Gag antigen, we initially conducted a dose-dependent study to determine the optimum AdVCA0848 dose that would significantly modulate adaptive immunity specific to the co-injected 5x106 vps/mouse dose of AdGag. 6-8 weeks old male BALB/c mice (n= 4) were intramuscularly (i.m.) co-injected in the tibialis anterior with viral particles in a phosphate-buffered saline solution in 30 I (PBS, pH 7. 4) containing a dose of 5x106 vps of AdGag along with 3 different doses of 5x 107, 5x108, or 5 x 109 vps/mouse of either AdNull or AdVCA0848. An additional group of mice were not injected (naive).
Additional experiments were conducted in which mice were co-injected with AdGag at 5x106vps/mouse and 5 x 109 vps/mouse of AdVCA0848 or AdVCA0848'd, or not injected (naives). At 14 dpi, mice were sacrificed, peripheral blood samples collected and spleen was harvested in 2% FBS media. To determine the effect of AdVCA0848 on the adaptive immune response against C. difficile-derived Toxin B antigen, female 6-8 weeks old C57BL/6 mice (n=4) were i.m. co-immunized in the tibialis anterior with viral particles of AdTox13 (5 x108 vps/mouse) along with 5x108 vps/mouse of either AdGFP or AdVCA0848.
At 21 dpi, mice were terminally sacrificed, and blood samples were collected for B cell analysis with ELISA. To verify the expression of Gag protein in the injected mice, 6-8 weeks old male BALB/c mice were iv. injected with 1x1011vps/mouse of AdGag only (n=3), or co-injected of lx1011 vps/mouse of AdGag along with lx1011 vps/mouse of either AdNull or AdVCA0848. At nearly 24 hpi, mice were humanely sacrificed and liver samples were obtained and frozen at -80 C until analysis by western blot for Gag protein levels.
3. Quantification of in vivo c-di-GMP synthesis Liver samples were harvested from mice injected with 2x109vps/mouse AdVCA0848, or 2 x10" vps/mouse of AdVCA0848, AdVCA0848'`, AdVCA0956, AdNull, or not injected (naives) as described in the animal procedures. 20 mg from each liver sample was placed in 500 L PBS and homogenized using an Omni Tissue Homogenizer (Omni International). 300 L of homogenate was added to an equal volume of equilibrated Phenol Solution (Sigma-Aldrich, St. Louis, MO). The homogenate-phenol solution was then vortexed and centrifuged at 15,000 rpm for 10 minutes. The aqueous phase was removed and added to 500 L chloroform. The mixture was vortexed and then centrifuged at 15,000 rpm for 10 minutes. The aqueous phase was removed and stored at -80 C until analysis. Quantification of c-di-GMP was conducted by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) at Michigan State University spectrometry & metabolomics core facility as previously described (Massie, JP
et al. (2012) Proc. Nall Acad Sei USA 109: 12746-12751).
4. Western blot for Gag protein Liver samples from mice injected with AdGag alone, or co-injected with AdGag and AdNull or AdVCA0848 as described above were harvested, and later were homogenized in ice cold lysis buffer containing 1% Triton and complete protease Inhibitor.
Supernatant was collected and analyzed for protein concentration (BCA protein kit; Sigma-Aldrich, St. Louis, MO). Total protein of 15 pg was heated at 100 C for 5 min with Laemmli sample buffer (Sigma Aldrich, St. Louis, MO), and samples were loaded on 1 mm-thick 10% gel Mini-Protean TGX Precast Gels (BIO-RAD, Hercules, CA, USA).
Transfer was completed overnight at 4 C using a 0.2 urn Nitrocellulose membrane (Millipore, Billerica, MA). The membrane was blocked for 1 h in Odyssey Blocking Buffer (Licor Biosciences - U.S., Lincoln, NE), then incubated for 1 hour at room temperature with primary monoclonal mouse anti Gag (1:10,000) antibody (183-H12-5C) obtained from the NTH-AIDS research and reference reagent program (gift from Dr. Y-H
Zheng, Michigan State University), and mouse anti-13-actin (1:3000) (#8224;
Abcam, Cambridge, MA) diluted in Odyssey Blocking Buffer (#927-40000, Licor, Lincoln, NE).
The blot was washed with TBS-T three times, and then incubated with labeled anti-mouse secondary antibody (#926-32210; Licor, Lincoln, NE) diluted in blocking buffer (1:10,000) for 1 hour at room temperature. The blotted membrane was washed and developed on the Licor Odyssey (Licor, Lincoln, NE).
5. ELISA
Effects of AdVCA0848 on IFN-13 induction was determined by quantifying IFN-13 using the VeriKineTM mouse IFN-13 ELISA kit (PBL Assay Science, Piscataway, NJ) according to the manufacturer's instructions. To determine the effect of AdVCA0848 on B
cell adaptive immune responses specific to antigens delivered by the co-administered AdGag or AdToxB, or the extracellular antigen OVA with the use of AdNull or AdVCA0848't as a negative control, ELISA-based titering experiments were conducted as previously described (Appledorn, DM etal. (2011) Clin Vaccine Immunol 18: 150-160).
Briefly, 5x 108 vps/well of inactivated Ad5 particles, 0.2 mg/well of Gag protein, 50 pg,/well of OVA, or 100 ng/well of ToxB (each diluted in PBS) was used to coat wells of a 96-well plate overnight at 4 C. Plates were washed with PBS-Tween 20 (0.05%) solution, and blocking buffer (3% BSA in PBS) was added to each well and incubated for 1-3 h at room temperature. For measuring total IgG Abs, plasma from injected mice was serially diluted in PBS buffer. Following dilution, plasma was added to the wells and incubated at room temperature for 1 h. Wells were washed using PBS-Tween 20 (0.05%), and HRP-conjugated rabbit anti-mouse Ab (Bio-Rad, Hercules, CA) was added at a 1:5000 dilution in PBS-Tween 20. Tetramethylbenzidine (Sigma-Aldrich, St. Louis, MO) substrate was added to each well, and the reaction was stopped with 2 N sulfuric acid.
Optical density (0.D.) was then obtained by reading the plates at 450 nm in a microplate spectrophotometer.
6. ELISPOT

Splenocytes were harvested from individual mice and red blood cells were lysed using ACK lysis buffer (Invitrogen, Grand Island, NY). Ninety-six-well Multi-Screen high protein binding Immobilon-P membrane plates (Millipore, Billerica, MA) were wetted with 70% ethanol, coated with mouse anti¨IFN-y or IL-2 capture Abs, incubated overnight, and blocked prior to the addition of 5x105 (AdGag studies) or lx106(OVA studies) splenocytes/well. Additional studies were conducted using AdVCA0848't as a control (AdGag studies) with the use of lx106splenocytes/well. Ex vivo stimulation included incubation of splenocytes in 100 IA media alone (unstimulated) or media containing 4 g/m1 Gag-specific AMQMLKETI (AMQ) peptide (GenScript, Piscataway, NJ) for the AdVCA0848 and AdGag studies, or 10 gg/m1 OVA or SIINFEKL (MHC class I-restricted OVA-derived peptide (Ahlen, G et al. (2012) PLoS One 7: e46959)) for AdVCA0848 and OVA studies, overnight in a 37 C, 5% CO2 incubator. Staining of plates was completed per the manufacturer's protocol. Spots were counted and photographed by an automated ELISPOT reader system (Cellular Technology, Cleveland, OH). Ready-SET-Go! IFN-y and IL-2 mouse ELISPOT kits were purchased from eBioscience (San Diego, CA).
7. Flow Cvtometry Analysis To investigate innate immune responses following AdVCA0848 vaccination, mice were injected with lx101 vps/mouse of AdVCA0848 vector and activation of innate immune cells was evaluated 6 hours following i.v. injection. Splenocytes were stained with various combinations of the following antibodies: PE-CD69 (clone: Hi .2F3), allophycocyanin-Cy7-CD3 (clone: 145-2C11), PerCP-Cy5.5-CD19 (clone: 1D3), Pacific Blue-CD8a (clone: 53-6.7), and PE-Cy7-NK1.1 (clone: PK136) (4 ig/m1). To assess the effect of AdVCA0848 on dendritic cells (DCs), splenocytes were stained with combinations of the following antibodies: PE-Cy7-CD11 c (clone: HL3), allophycocyanin (APC)-Cy7-CD1lb (clone: M1/70), Alexa Fluor 700-CD8a (clone: 53-6.7), FITC-CD40 (clone:

3), PerCP-Cy5.5-CD80 (clone: 16-10A1), and V450-CD86 (clone: GL1) (4 1.1g/m1).
All antibodies were obtained from BD Biosciences. To determine the intracellular cytokine levels 14 dpi of AdVCA0848 and AdGag co-injections, intracellular staining was performed as previously described (Aldhamen, YA etal. (2012)J Immunol 189:

1359). Briefly, splenocytes (2.5 x106/well) were stimulated with Gag-specific AMQ peptide for 6 hours with Brefeldin A (BFA) (Sigma-Aldrich, St. Louis, MO) for 30 minutes and stored at 4 C overnight. Cells were washed twice with FACS buffer and surface stained with APC-CD3, Alexa Fluor 700-CD8a, and CD16/32 Fc-block Abs, fixed with 2%

formaldehyde (Polysciences, Warrington, PA), permeabilized with 0.2% saponin (Sigma-Aldrich, St. Louis, MO), and stained for intracellular cytokines with PE-Cy7-TNF-a, and Alexa Fluor 488-IFN-y (4 g/ml) (all obtained from BD Biosciences, San Diego, CA). We included a violet fluorescent reactive dye (ViViD; Invitrogen) as a viability marker to exclude dead cells from the analysis. Tetramer staining of splenocytes at 1x106 cell/well was performed using PE-labeled MHC class I tetramer folded with the AMQ
peptide (generated at the NIH Tetramer Core Facility (Atlanta, GA)) for 30 minutes at room temperature, and for memory T cell staining, a mixture of the following antibodies (at 2 ug/m1) were used: APC-CD3, Alexa Fluor 700-CD8a, PerCP-Cy5.5-CD127, FITC-CD62L, and CD16/32 Fe-block Abs. All antibodies were purchased from BD Biosciences (San Diego, CA). After washing with FACS buffer, data for stained cells were collected with the use of BD LSR 11 instrument and analyzed using FlowJo software (Tree Star, San Carlos, CA). Gating strategy was based on negative control results (naives) that were applied consistently across all samples examined. Representative examples from this gating approach are presented here for activation of innate immunity cells and for the frequency of cytokine-producing CD8+ T cells.
8. Statistical analysis Statistically significant differences in innate immune responses were determined using a one-way ANOVA with a Student¨Newman¨Keuls post hoc test (p value of <0.05 was deemed statistically significant). The ELISPOT and ELISA studies were all analyzed using one-way ANOVA with a Student¨Newman¨Keuls post hoc test (p value of <0.05 was deemed statistically significant). For flow cytometry, a one-way ANOVA with a Student¨
Newman¨Keuls post hoc test was used (p value of <0.05 was deemed statistically significant). Statistical analyses were performed using GraphPad Prism (GraphPad Software).
Example 7: AdVCA0848 produces significant amounts of c-di-GMP in vivo in mice Examples 1-5 above demonstrated the feasibility of in vitro and in vivo production of c-di-GINTP in mammalian cells by using Ad5 vectors to transduce DGCs. Prior unpublished studies by the inventors suggested that use of an alternative DGC, VCA0848, which has greater enzymatic activities, might generate a significantly elevated amount of c-di-GMP in vivo. An Ad5 vector with a CMV enhancer/promoter element to drive expression in mammalian cells was constructed. The use of the AdVCA0848 platform resulted in a significant in vivo c-di-GMP production measured in the liver of injected mice.

Injecting with increasing viral loads of 2x109vps/mouse and 2x1011 vps/mouse of AdVCA0848 resulted in approximately 130 timol/g and 3000 umol/g c-di-GMEP in the liver, respectively. This confirms that the in vivo c-di-GMP production is entirely due to the enzymatic activity of the delivered VCA0848 as AdVCA0848'1vectors and nave mice failed to produce detectable levels of c-di-GMP (Figure 9). Additionally, when compared to an earlier DGC-expressing platform that was constructed using the exact same adenovirus vector backbone, the AdVCA0848 platform produces significantly higher levels of c-di-GMP in the mouse liver (¨ 400-fold increase) than that produced by an equal viral dose of the AdVCA0956 platform per gram of mouse liver (p<0.05). As expected, similar to AdVCA0848'1control, the AdNull vectors, which lack the DGC gene, did not produce detectable levels of c-di-GMP (Figure 17). These results confirm the feasibility of transducing the bacterial DGC VCA0848 using Ad5 to synthesize in vivo larger amounts of c-di-GMP in vivo.
Example 8: AdVCA0848 activates innate immune responses It was thought that activation of beneficial innate immune responses by adjuvants is the underlying mechanism that is critical for achieving effective and long-lived, antigen-specific, adaptive immune responses. Intravenous administration of AdVCA0848 dramatically induced plasma levels of IFN-f3 (p<0.05) nearly 1000-fold compared to the level produced by the AdNull control (Figure 10A). Importantly, administration of AdVCA08481"1 control produced similar levels of IFN-r3, as compared to AdNull, suggesting the increased IFN-f3 levels following AdVCA0848 is due to the enzymatic activity of the transduced VCA08484 (Figure 18A). Also, administration of AdVCA0848 significantly induced DC maturation and NK activation as compared to an identical cell population derived from AdNull controls (p<0.05) (Figures 10B 84 10C).
Furthermore, administration of AdVCA0848 resulted in increased numbers of CD69-expressing B
cells, CD3+CD8- and CD3+CD8+ T cells, as compared to the use of the AdNull vector in this experiment (p<0.05) (Figures 10D-10F). Utilization of AdVCA0848mut control suggested that the activation of immune cells is largely due to the enzymatic activity of the transduced VCA0848 (Figures 18B-18F). Our results also confirmed previous findings that the Ad5 vector itself results in increased activation of NK cells, macrophages, CD3+CD8" T cells, CD3+CD8+ T cells, and B cells as indicated by the significant expression of the activation marker CD69 (Aldhamen, YA et al. (2012)J Immunol 189: 1349-1359). Together, these data suggest a significant induction of innate immune responses by AdVCA0848 in the mouse model, surpassing that caused by the adenovirus itself.
Example 9: AdVCA0848 enhances induction of antigen-specific adaptive T cell immune responses Direct administration of the ovalbumin (OVA) protein is a model antigen frequently used to study antigen-specific adaptive immune responses (Basto, AP et al.
(2015) Mol Immunol 64: 36-45; Garulli, B etal. (2008) Clin Vaccine Immunol 15: 1497-1504).
C57BL/6 mice were vaccinated with 100 pg/mL OVA alone, or simultaneously with AdNull or AdVCA0848; and a fourth untreated group served as a naïve control.
At 14 dpi, IFN-y ELISPOT results from the experimental and control animals indicated that OVA-specific T cell responses from mice co-administered with AdVCA0848 and OVA
were significantly higher (upon ex vivo stimulation with the entire OVA protein or the OVA-derived MHC class I-restricted peptide SIINFEKL) as compared to splenocytes derived from mice receiving only OVA, or OVA concomitant with the AdNull control vector (p<0.05) (Figure 11A). The simultaneous use of AdVCA0848 with OVA vaccination also increased the number of SIINFEKL and the intact OVA protein-specific IL-2-secreting T
cells present in the splenocytes of OVA-treated mice as compared to mice injected with OVA alone, or concomitant with AdNull control (p<0.05) (Figure 11C). The noticeable variability of T cell responses resulted from the ex vivo stimulation with whole OVA
protein and the MHC class I-restricted SIINFEKL peptide likely suggest a CD8+
T cell-driven response indicated by higher SIINFEKL-specific IFN-y producing T cells and smaller SIINFEKL-specific IL-2 producing T cells. Interestingly, splenocytes harvested from mice co-injected with AdVCA0848 and OVA also had dramatically increased numbers of Ad5 capsid-specific IFN-y-secreting T cells and 1L-2 secreting T
cells, as compared to mice injected with OVA alone, or concomitant with AdNull control (p<0.05) (Figures 11B and 11D). These results indicate that AdVCA0848 provides enhancement of OVA-specific adaptive T cell immune responses when co-injected with the extracellular antigen OVA.
Example 10: AdVCA0848 enhances induction of antigen-specific adaptive B cell immune responses Co-administering AdVCA0848 and OVA also resulted in enhancement of OVA-specific (Figure 12A) and Ad5-specific (Figure 12B) B cell responses 6 dpi. At 14 dpi, OVA-specific B cell response was enhanced compared to mice co-injected with the AdNull control vector (Figure 12C) or when injected with OVA alone (p<0.05) (Figure 19). Ad5-specific IgG antibody B cell responses were also detected in those mice that received either of the Ad5 vectors. While the presence of AdVCA0848 significantly increased the Ad5-specific B cell response compared to that exerted by the AdNull control (p<0.05) when measured at 6 dpi, this effect was observed to be minimal when measured at 14 dpi (Figure 12D). Despite the transient enhancement of humoral response against the delivering vector, these results demonstrate the beneficial effects of AdVCA0848 on the OVA-specific adaptive B cell response from a single administration of OVA.
Example 11: Sustained high-level production of c-di-GMP can inhibit T cell responses to antigens expressed from viral vectors The previous results indicated a modest, although significant, enhancement of adaptive immune responses specific against antigens expressed from Ad5-based vaccines co-injected with AdVCA0956, a vector expressing a less active DGC (Examples 1-5).
Therefore, it was assessed whether the enhanced ability of AdVCA0848 to produce c-di-GMP in vivo would also improve adaptive immune responses specific for adenovirus-expressed antigens. An adenovirus-based vector was previously used to express the Gag protein, an HIV-1-derived antigen, and demonstrated the platform's ability to induce Gag-specific humoral and cellular immune responses (Aldhamen, YA et al. (2011)J
Immunot 186: 722-732; Appledorn, DM etal. (2010) PLoS One 5: e9579; Appledorn, DM et al.
(2011) Clin Vaccine Immunol 18: 150-160; Gabitzsch, ES et al. (2009) Immunol Lett 122:
44-51). Based on the previous work, the AdGag vaccine was administered at the dose of 5x106 vps/mouse along with escalating doses (5 x 107, 5 x 108, or 5 x 109 vps/mouse) of AdVCA0848 or the AdNull control. After 14 days, Gag-specific memory T cell immune responses were evaluated by IFN-y ELISPOT assay. The results demonstrated that concurrent administration of AdVCA0848 along with the AdGag vaccine inhibited T cell responses to the Gag antigen, which were especially significant at the highest AdVCA0848 dose of 5x 109 vps/mouse compared to that seen from the concurrent administration of AdNull control along with AdGag vaccine (p<0.05) (Figure 13A). Similar to the previous observations (Schuldt, NJ etal. (2011) PLoS One 6: e24147), as the viral load of AdNull co-injected with AdGag increased, the Gag-specific T cell response measured by IFN-y ELISPOT decreased in a dose-dependent manner (p<0.05). In contrast, ELISPOT
assays demonstrated a dramatic enhancement of Ad5-specific IFN-y-producing T cells at 5x109 vps/mouse of AdVCA0848 compared to the AdNull control group (p<0.05), while the first two doses of 5x107 and 5x 108vps/mouse showed minimal Ad5-specific T cell response (Figure 13B) It was confirmed that the inhibitory effects on IFN-y-secreting T
cells was lost in a VCA0848 mutant that cannot synthesize c-di-GMP (Figure 20A).
A multi-parameter tetramer-binding assay showed a significantly decreased number of Gag-specific Tet+CD8+ T cells present in mice co-injected with three different doses of AdVCA0848 along with AdGag as compared to mice co-injected with AdGag and the AdNull control vector (p<0.05) (Figure 14A), confirming the negative impact of AdVCA0848 on the induction of Gag-specific CD8+ T cells. Intracellular staining (ICS) and FACS analysis was also performed to evaluate the impact of AdVCA0848 on the numbers of Gag-specific CD8+ T cells upon ex vivo stimulation with the Gag-specific peptide, AMQ.
The number of IFN-y and TNF-a-producing CD8+ T cells specific for this potent Gag peptide were significantly inhibited in mice co-injected with AdVCA0848 as compared to equal viral loads of AdNull (p<0.05) with the highest dose of AdVCA0848 of 5 x109 vps/mouse showing the strongest inhibitory effects (Figures 14B & 14C) The effect of AdVCA0848 on Gag-specific IFN-y, INF-a and IL-2-producing CD4+ T cells was also looked at and no significant effect was observed (data not shown). Together, these data strongly suggested that despite a strong induction of innate immunity, and improved induction of adaptive immune responses to extracellular proteins such as the OVA protein and the Ad5 capsid, expressing high levels of c-di-GMP using VCA0848 from an Ad5 vector significantly inhibited induction of antigen specific CD8+ T cell responses to antigens expressed intracellularly by another Ad5 vector.
Example 12: Sustained high-level production of c-di-GMP can also inhibit B
cell responses to antigens expressed from viral vectors Humoral B cell responses following AdVCA0848 co-administration with AdGag were evaluated. Similar to its effect on T cell responses, the presence of AdVCA0848 resulted in significant inhibition of HIV-1/Gag-specific B cell responses as compared to those mice administered with equal amounts of the AdNull control vector (p<0.05) (Figure 15A). The inhibition of Gag-specific B cell responses by AdVCA0848 was very potent at the doses of 5 x107 and 5 x108 vps/mouse (compared to AdNull, p<0.05). AdNull exhibited inhibition similar to AdVCA0848 at the highest dose of 5 x109 vps/mouse (Figure 15A).
Alternatively, increasing doses of both the AdNull and AdVCA0848 increased B
cell responses against the Ad5 vector in a dose-dependent manner (Figure 15B). The inhibitory effects on Gag-specific B cell responses were lost using the AdVCA08481"1 that cannot synthesize c-di-GMP (Figure 20B). The ability of AdVCA0848 to enhance Ad5-specific B
cell response compared to that shown by AdVCA0848 mut was confirmed (Figure 20C).
To confirm this interesting observation using a different antigen expressed by an Ad5-based vaccine, we co-administered AdVCA0848 along with an Ad5 vector expressing the truncated form of the C. difficile-derived Toxin B protein (AdToxB). The presence of AdVCA0848 with AdToxB also resulted in significantly reduced ToxB-specific B
cell responses as compared to control vaccinations (p<0.001) (Figure 15C).
Importantly, significantly (p<0.01) increased Ad5-specific IgG titers in mice vaccinated with AdVCA0848 and AdToxB was again obsereved, as compared to controls (Figure 15D).
These results further confirm the inhibitory effects of the strong c-di-GMP
producer, AdVCA0848, on another antigen intracellularly expressed from an adenovirus vector (AdToxB).
Example 13: Co-administration of AdGag and AdVCA0848 doesn't inhibit Gag expression One possible explanation for the inhibition of response to Ad-expressed antigens is that the presence of the AdVCA0848 vector inhibits in trans the in vivo expression of the Ad expressed antigens. However, mice co-injected with AdVCA0848 and AdGag demonstrated the presence of the HIV-1 derived Gag protein whether delivered by the AdGag platform alone, or when co-injected with the AdNull control, or with AdVCA0848, (Figure 16). These results suggest that inhibitory effects exerted by AdVCA0848 on B cell and T cell adaptive immune responses against Gag are not due to lack of Gag expression and translation in vivo.
Discussion Understanding the molecular mechanisms underlying how a putative adjuvant acts to enhance the efficacy of a specific vaccine will help to guide the formulation of newer generation vaccines that efficiently generate specific long-term immunity against difficult antigens derived from pathogens or cancer cells (Rueckert, C etal. (2012) PLoS
Pathog 8:
e1003001). The use of pure c-di-GMP has been demonstrated to be an immune-modulatory molecule with potential therapeutic and prophylactic properties (Karaolis, D
K. et al.
(2007)J Immunol 178: 2171-2181). While the presence of nucleic acids can be sensed by AIM2, and signals the activation of caspase-1 (Hornung, V etal. (2009) Nature 458: 514-518; Femandes-Alnemri, T etal. (2009) Nature 458: 509-513), the presence of cytosolic c-.
di-GMP can be sensed by other sensors including the STING and helicase DDX41 pathways, and subsequently lead to the release of IFN-13, primarily from CD11b+ DCs (Huang, L etal. (2013)J Immunol 191: 3509-3513). Additionally, c-di-GMP has been shown to stimulate the MYPS/STING-dependent induction of INF-a and IL-22, not type I
IFN, when used as a nasal mucosal adjuvant, suggesting c-di-GMP may have different effects on different innate immunity pathways (Blaauboer, SM et al. (2014) J
Immunol 192:
492-502; Blaauboer, SM et al. (2015) eLife 4).
In this study, the ability of a potent, bacterial derived DGC to be delivered by an Ad5 vector (AdVCA0848) that produced more than 400-fold more c-di-GMP than the Ad5 DGC vector described above (Examples 1-5) was demonstrated, resulting in a robust induction of several innate immune responses, including 1FN-13 induction. By using a mutant version of VCA0848 delivered by AdVCA0848't, the data herein suggests that these significant levels of c-di-GMP are products of the enzymatic activity of the transduced VCA0848. These strong innate immune responses allowed the induction of enhanced adaptive immune responses to an extracellular antigen, i.e. OVA, co-administered with the AdVCA0848, but also suppressed adaptive immune responses to virally expressed antigens. The recent characterization of mammalian endogenous cyclic GMP-AMP
(2'3'-cGAMP) synthetase (cGAS) (Wu, Jet al. (2013) Science 339: 826-830; Ablasser, Act al.
(2013) Nature 503: 530-534; Zhang, X et al. (2013)Mol Cell 51: 226-235) provided the rationale for testing cGAMP as a vaccine adjuvant, and initial studies demonstrated its usefulness in stimulating innate immune responses and improving antigen-specific adaptive immune responses (Li, XD et al. (2013) Science 341: 1390-1394; Gao, D etal.
(2013) Science 341: 903-906; Skrnjug, I etal. (2014) PLoS One 9: el10150). When compared to the bacterial c-di-GMP, cGAMP had higher binding affinity to STING. However, it has also been shown that c-di-GMP results in higher IFN-13 induction than that induced by 2'3'-cGAMP or its isomers, suggesting that higher binding affinity to STING does not correlate with IFN-13 induction. These results may be attributable to possible differences in biological stability between c-di-GMP and the mammalian cGAMP (Zhang, X etal.
(2013) Mol Cell 51: 226-235).
The adenovirus-based platforms utilized in the present studies described herein are also expected to activate multiple innate immune responses. The vector is known to activate =

innate immune responses via interactions with extracellular and intracellular TLRs, and can simultaneously trigger early pro-inflammatory responses such as the induction of IP-10 (Tibbles, L A. et al. (2002)J Virol 76: 1559-1568) and the activation of the P13K signaling cascade (Verdino, P et al. (2010) Science 329: 1210-1214). It has been also demonstrated that upon penetrating host cells and escaping the endosomal compartment, adenoviral vectors have the ability to ignite the MAPK and NFKI3 signaling pathways through TLR-dependent (TLR2, 3, 4, and 9) and non-TLR dependent mechanisms (Appledorn, DM
et al.
(2008)J Immunol 181: 2134-2144; Zhu, J et al. (2007)J Viral 81: 3170-3180;
Appledorn, DM et al. (2009)J Innate Immun 1: 376-388) leading to the induction of several chemokines and cytokines, fostering its utility as a vaccine platform in and of itself.
Additionally, the adenoviral dsDNA genome can be sensed by cytoplasmic sensors such as DAI (leading to type I IFN induction) (Ishii, KJ et al. (2008) Nature 451: 725-729) and AIM-2 resulting in activating the inflammasome and the induction of caspase-l-dependent IL-113 (Hornung, V etal. (2009) Nature 458: 514-518). Recent data also suggest that STING is central and acts as a major PRR after vaccination with Ad5-based platforms including Ad5 vectors (Quinn, KM et al. (2015)J Clin Invest 125: 1129-1146).
With these facts in mind, it is clear that these results confirm that the additional production of c-di-GMP from an already immunogenic platform such as Ad is significant enough to further promote the induction of pro-inflammatory immune responses beyond that provided by the Ad vector platform itself. Whether expression of DGCs from other vaccine platforms will yield similar results awaits future studies beyond the scope of this manuscript.
The broad impact of the AdVCA0848 platform on innate immune responses clearly demonstrates its promising potential for use as a vaccine adjuvant to enhance adaptive immune responses. For example, relative to enhancing adaptive immune responses to extracellular antigens, plasmacytoid dendritic cell precursors (pDC) are thought to be the major source of IFN-i3 (Soumelis, V etal. (2006) Eur J Immunol 36: 2286-2292).
In agreement with previous reports that demonstrated the stimulatory effects of c-di-GMP on murine and human DCs (Elahi, S etal. (2014) PLoS One 9: e109778; Karaolis, D
K. et al.
(2007)J Immunol 178: 2171-2181), AdVCA0848 improved the induction of CD11c CD1lb" CD86+ Des. Ultimately, pDCs can differentiate into typical DCs capable of stimulating naive T cells in an antigen-specific manner (Renneson, J etal.
(2005) Clinical and experimental immunology 139: 468-475). IFN-13 has also been shown to enhance DC
maturation, the efficiency of DC's to activate the cross-priming of CD8+ T
cells, and increase induction of CD4+ Th I differentiation (Huber, JP etal. (2011) Immunology 132:
466-474). In addition to increasing the number of CD86+CD11c+CD11b-DCs and activating CD69+NK1.1*NK cells that are involved in regulating innate immune responses, AdVCA0848 activated cells directly involved in adaptive immune responses such as B cells and CD4+ and CD8+ T cells.
AdVCA0848 also enhanced induction of OVA-specific B cell and T cell adaptive responses. These results parallel recent studies evaluating the beneficial effects of direct administration of c-di-GMP as an adjuvant during vaccination with OVA
(Blaauboer, SM
etal. (2014)J Immunol 192: 492-502; Wu, J et al. (2013) Science 339: 826-830), and 4-Hydroxy-3-nitrophenylacetyl-Chicken Gamma Globulin, NP-CGG, in which c-di-GMP
was shown to have the capacity to enhance germinal center (GC) development (Gray, PM
et al. (2012) Cell Immunol 278: 113-119). Additionally, the presence of c-di-GMP in an adjuvant formulation containing chitosan (CSN) improved adaptive immune responses to H5N1 antigens (Svindland, SC etal. (2013) Influenza Other Respir Viruses 7:
1181-1193), and (along with a conventional aluminum salt-based adjuvant) improved adaptive immune responses specific to the hepatitis B surface antigen (HBsAg) (Gray, PM etal.
(2012) Cell Immunol 278: 113-119). Recently, it was demonstrated that nasal administration of c-di-GMP significantly increases the MYPS-mediated uptake of OVA antigen via endocytosis and pinocytosis in vivo. This generates mucosal adjuvant activities that are mediated by type II and type III interferon but not type I interferon suggesting variable c-di-GMP
pleiotropic effects on innate immune responses against extracellular antigens.
The in vivo .
production of c-di-GMP by i.m. administration of our AdVCA0848 platform potentially enhanced the OVA uptake and processing by DCs, and subsequently resulted in improved OVA-specific adaptive immune responses (Blaauboer, SM et al. (2015) eLife 4).
As a proof of principle, our results suggest that adenovirus-based platforms expressing DGCs may also be used to promote improved immunity against other disease specific antigens, such as those found in current cholera, diphtheria, and tetanus vaccines, as each are examples of protein-based vaccines In addition, as our approach also enhances activation of antigen-presenting cells (APCs) and induction of antigen CD8+ cytotoxic T lymphocytes (CTLs), future studies using tumor antigen specific peptides may also enhance the induction of anti-tumor cellular immune responses (Miyabe, H etal. (2014) J Control Release 184:
20-27;
Chandra, D et al. (2014) Cancer Immunol Res 2: 901-910; Karaolis, DK et al.
(2005) Biochem Biophys Res Commun 329: 40-45; Joshi, VB et al. (2014) Expert review of vaccines 13: 9-15).
The results described herein also revealed the potential for inhibitory effects on adaptive immune responses to antigens expressed intracellularly, simultaneous with provision of high levels of c-di-GMP. Although, the dose of 5X 108 vps/mouse of AdVCA0848 did not show significant inhibition of 1FN-y-secreting splenocytes compared to that shown by the AdNull control, this dose caused significant inhibition of Gag-specific 1FN-y and TNF-a-secreting CD84-T cells, suggesting that CD8+ T cells may be the specific targets for these inhibitory effects. Furthermore, increasing the AdVCA0848 dose to 5 X109 vps/mouse further inhibited Gag-specific T cell responses. Of note, the use of higher doses of the AdNull control vector also resulted in decreased induction of Gag-specific CD8+ T
cell responses. Despite this, the provision of elevated c-di-GMP levels resulted in additional inhibitory effects on Gag-specific adaptive immune responses.
Examples 1-5 show that increasing the dose of AdVCA0956 to 5 x109 vps/mouse did not improve B cell responses specific for an antigen delivered by an Ad5 vector in mice (Examples 1-5). Specifically, AdVCA0956 moderately suppressed B cell responses against the C. clifficile-derived Toxin A antigen expressed from the co-injected Ad5 vector at the dose of 5x 109 vps/mouse. The results herein suggest that those trends were likely real. Even stronger inhibitory effects were noted after administration of the more potent AdVCA0848 on B cell and T cell adaptive immune responses against the intracellularly expressed Gag and ToxB antigens. These results suggest that in mice the magnitude of inhibitory effects on adaptive immune responses to intracellularly expressed antigens is likely to increase with excessive amounts of c-di-GMP production.
There is also the possibility that the transduced DGC, and ultimately the synthesized c-di-GMP, interferes with the expression of these antigens when using the CMV
expression cassette (used in constructing the vectors). This possibility was explored in vitro herein, and found enhanced GFP expression in 1-1EK293 cells co-infected with AdVCA0848 and an Ad5 vector expressing GFP (AdGFP) from the same CMV enhancer/promoter elements used in these studies (data not shown). These data also suggest that co-administration of the AdGag vaccine along with the strong c-di-GMP producing AdVCA0848 did not prevent Gag translation. It remains unclear how the significant induction of c-di-GMP
and subsequently high levels of type I IFN can inhibit the T cell and B cell responses of an intracellularly expressed antigen (Quinn, KM etal. (2015) J Clin Invest 125:
1129-1146), and the impact of strong type I IFN induction on the availability of intracellular antigen-loaded APCs requires further investigation. It is noted that the production of another bacterial second messenger, c-di-AMP, by the intracellular pathogen Listeria monocytogenes was shown to induce IFN-I3 in a STING-dependent manner leading to the inhibition of T cell-mediated immunity, similar to our results with excessive production of c-di-GMP (Archer, KA et al. (2014) PLoS Pathog 10: e1003861).
In summary, demonstrated herein is the feasibility of in vivo synthesis of extremely large amounts of c-di-GMP via an Ad5-based platform expressing a highly potent DGC.
While high amounts of c-di-GMP production can inhibit adaptive immune responses to antigens expressed simultaneously with significant increasing c-di-GMP levels, this unique platform appears to preferentially improve antigen specific B cell and T cell adaptive immune responses specific for co-administered extracellular antigens. This approach can be utilized to develop and improve protein-based prophylactic and therapeutic vaccines targeting infectious diseases and cancers.
Incorporation by Reference The contents of all references, patent applications, patents, and published patent applications, as well as the Figures and the Sequence Listing, cited throughout this application are hereby incorporated by reference.
Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the following claims.

=

SEQUENCE LISTING
<110> BOARD OF TRUSTEES OF MICHIGAN STATE UNIVERSITY
<120> COMPOSITIONS AND METHODS FOR INDUCING AND ENHANCING AN IMMUNE
RESPONSE AGAINST INFECTIONS, DISEASES, AND DISORDERS
<130> P11918 <140> PCT/US2016/052198 <141> 2016-09-16 <150> US 62/219,387 <151> 2015-09-16 <160> 170 <170> ASCII TEXT
<210> 1 <211> 1884 <212> DNA
<213> Vibrio cholerae <400> 1 tcacgcaaag tgatgcattt ccatggcggt gagtactgat atttggttgc gtcccgatgt 60 tttggattca tataaagcca gatcggctct tttgtagctt tggtcgggtg atgtgcaaac 120 atcggtaaca ccaccactta gggtcacttg ttgatggtgt aatgaagcga tatgcaggcg 180 tacgcggtta agtacttgtt cggcttcttc aatggaagtg taggggaaaa taatggcaaa 240 ctcttctccg ccaatccgtg cgataaaatc cgattcccgt aactgatctt ggatgccttt 300 cgcaacggtc cgtaacacta ggtccccttc gttgtgtccg aatttgtcgt taatgcgttt 360 aaagtggtcg atatcaatga tagcaaggca gctctgggct tgatcgggat aacggcgacg 420 cttagcgcac tctaaagaga tggtttgatc gaatttacgt cgattccaca aatcggttaa 480 cgcatctttt tcgctcagct cacgcaggcg attctccagc gccttgcgat gtgaaatatc 540 cacaaaagag gcaacgtaga attgaatgac attgtcttca tcgcggatgc tttgaatacg 600 gagaatttcg gtgatgcttt cgccatcttt gcgtttgttg atcacttcac cttcccatac 660 gccattgtct tgcagagctt tccacatctg catatagaat tcgactttgt gtaatccaga 720 agcaaaaatg gacggctgct tacctttgac atcttcaaaa gtgtaaccac ttaggcgggt 780 aaattcgttg tttactttga tgatgcgatt ctggcggtcg gtaatgacca ccgctgacat 840 gccatccatc gctgctcgag ccaatttact gtcaaggcta ttttttaaat ggttgatgtt 900 ccatgccgca aatccagccg caatgataga gagtagcgat aacactgtca ccgcttgact 960 catcagtgcc cagcgcgcat ttgcgtaggt cttatctatt tctgccttat tgatgcgcag 1020 taccaatacc aaaggtttaa agtcaggtaa gacagaactg agatccactt tgatatagct 1080 aaaccaggtt tgattggata gagcaaagcc ttgttggttg agttggattt tttgccaaag 1140 ctctgggtgt tgggctgaaa agtggagtga agaggttgaa cgtgtaccgg atggcttgtg 1200 ttcactgagc agtaattctc ctgccgaatt caaaatatcc ggtgaatcaa actgatcata 1260 aataaaagag agacgttgat agagagactg tagcttcacc gtcacgacaa gaaaaccttg 1320 =
ccgttggcct tgatgctcaa tacccgtcac aaaacgaaag gtcggcagca taccagaagg 1380 cgtatctgct gacatcgcga cttgcgttgc ccaaacttga ggcgtcgtga gttgggcgta 1440 ttgagccaca atttgctggc tgaacggatc tgtcgtttga gcagattcaa caaaggtgac 1500 ttggtgccca tcgtaaatcg ctttaagttg ttcttttcct tgtctatcca gcaatctgaa 1560 tgaagagaaa atcgcttgcg atcttaacgt cacatcccac aatgttttga gttgactgag 1620 tgcttctttg cttggtgtgg tgacagccgt gaataaaagg tcatttttag ctaacagctg 1680 ggtggcttgg tgtgtgcttt ccagcattcg taacaagtca tgctgactga actcaagctg 1740 taagcgagtc tgtttttcaa cgctgctgac cgcttgagtc tcaagctggc tagcagcatg 1800 tatgaaatac agtgtaggaa tgaaaccaag tacaaacgca acaatggcaa attgtatgaa 1860 atatttacgg gctgaggtgt acat 1884 <210> 2 <211> 627 <212> PRT
<213> Vibrio cholerae <400> 2 Met Tyr Thr Ser Ala Arg Lys Tyr Phe Ile Gin Phe Ala Ile Val Ala Phe val Leu Gly Phe Ile Pro Thr Leu Tyr Phe Ile His Ala Ala Ser Gin Leu Glu Thr Gin Ala Val Ser Ser val Glu Lys Gin Thr Arg Leu Gin Leu Glu Phe Ser Gin His Asp Leu Leu Arg Met Leu Glu Ser Thr His Gin Ala Thr Gin Leu Leu Ala Lys Asn Asp Leu Leu Phe Thr Ala Val Thr Thr Pro Ser Lys Glu Ala Leu Ser Gin Leu Lys Thr Leu Trp Asp val Thr Leu Arg Ser Gin Ala Ile Phe Ser Ser Phe Arg Leu Leu Asp Arg Gin Gly Lys Glu Gin Leu Lys Ala Ile Tyr Asp Gly His Gin val Thr Phe val Glu Ser Ala Gin Thr Thr Asp Pro Phe Ser Gin Gin Ile Val Ala Gin Tyr Ala Gin Leu Thr Thr Pro Gin Val Trp Ala Thr 4 .

1 .
Gin Val Ala met Ser Ala Asp Thr Pro Ser Gly Met Leu Pro Thr Phe Arg Phe Val Thr Gly Ile Glu His Gin Gly Gin Arg Gln Gly Phe Leu val val Thr val Lys Leu Gin Ser Leu Tyr Gin Arg Leu Ser Phe Ile Tyr Asp Gin Phe Asp Ser Pro Asp Ile Leu Asn Ser Ala Gly Glu Leu Leu Leu Ser Glu His Lys Pro Ser Gly Thr Arg Ser Thr Ser Ser Leu His Phe Ser Ala Gin His Pro Glu Leu Trp Gin Lys Ile Gin Leu Asn Gin Gin Gly Phe Ala Leu Ser Asn Gin Thr Trp Phe Ser Tyr Ile Lys 260 265 270 ' Val Asp Leu Ser Ser Val Leu Pro Asp Phe Lys Pro Leu Val Leu val Leu Arg Ile Asn Lys Ala Glu Ile Asp Lys Thr Tyr Ala Asn Ala Arg Trp Ala Leu Met Ser Gin Ala val Thr Val Leu Ser Leu Leu Ser Ile Ile Ala Ala Gly Phe Ala Ala Trp Asn Ile Asn His Leu Lys Asn Ser Leu Asp Ser Lys Leu Ala Arg Ala Ala met Asp Gly Met Ser Ala val Val Ile Thr Asp Arg Gin Asn Arg Ile Ile Lys Val Asn Asn Glu Phe Thr Arg Leu Ser Gly Tyr Thr Phe Glu Asp val Lys Gly Lys Gin Pro Ser Ile Phe Ala Ser Gly Leu His Lys val Glu Phe Tyr met Gin Met Trp Lys Ala Leu Gin Asp Asn Gly Val Trp Glu Gly Glu val Ile Asn Lys Arg Lys Asp Gly Glu Ser Ile Thr Glu Ile Leu Arg Ile Gin Ser . i Y .
Ile Arg Asp Glu Asp Asn Val Ile Gin Phe Tyr Val Ala Ser Phe Val Asp Ile Ser His Arg Lys Ala Leu Glu Asn Arg Leu Arg Glu Leu Ser Glu Lys Asp Ala Leu Thr Asp Leu Trp Asn Arg Arg Lys Phe Asp Gin Thr Ile Ser Leu Glu Cys Ala Lys Arg Arg Arg Tyr Pro Asp Gin Ala Gin Ser Cys Leu Ala Ile Ile Asp Ile Asp His Phe Lys Arg Ile Asn Asp Lys Phe Gly His Asn Glu Gly Asp Leu Val Leu Arg Thr Val Ala Lys Gly Ile Gin Asp Gin Leu Arg Glu Ser Asp Phe Ile Ala Arg Ile Gly Gly Glu Glu Phe Ala Ile Ile Phe Pro Tyr Thr Ser Ile Glu Glu Ala Glu Gin Val Leu Asn Arg Val Arg Leu His Ile Ala Ser Leu His His Gin Gin Val Thr Leu Ser Gly Gly Val Thr Asp Val Cys Thr Ser Pro Asp Gin Ser Tyr Lys Arg Ala Asp Leu Ala Leu Tyr Glu Ser Lys Thr Ser Gly Arg Asn Gin Ile Ser Val Leu Thr Ala Met Glu Met His His Phe Ala <210> 3 <211> 1386 <212> DNA
<213> Vibrio cholerae <400> 3 ttatgaccag gtacgaaaga caacctggtt ctttccattc cgctttcctt cgtacattaa 60 gctatcggca tcgtgcaaac tgaatggccg agctgggtgt aggtaaaatg cacagcctag 120 gctgatggtg agtgacagag agtgttgggc attcactacc cacttttttt ctgcaactcg 180 ttggcaaatt cgctcagcta actgctgcga ctcttctgca tttttaccac gcgctacaat 240 agcaaactct tcaccaccaa tccttgcaaa gtaggtatcc gatgctaaag cttgtcgcac 300 t .
acacccaacc acgaaacaga tggcattatc tcctgcgcca tgcccaaagc gatcgttaat 360 ggttttgaag tcatcaatat caaaaaccat caacgttaag ctgcctgatc gtgtttgttc 420 cgcttcaaga tgttcaaaaa acgaacggcg attagcaatg cccgtcaagc tatccgtttt 480 cgctaaatag gagagttttt gattggcttc ttcaagttgc tgtgttcgca atcgaacggt 540 acgccgtagc tgaagagtat aaataacgat actgagtaag agacctgaag cgagaatcgg 600 cattaagtaa cgtggataaa tcgtttcaat atgaacccat cgacttaaaa tacggttttt 660 ctcattgcta cttaattgtg caaacccctg ctctacttgc tctaataaat ccctattgcc 720 tttggcgacc gctggacgta attcctctga ataaagaaac ttcactggcg taaaatcttt 780 cgcgccattg gaaaccacta tatagaaatt ggcgacctga gtatcggcca caaaaccatc 840 taattctcgt cgctttgctg cagacatcat caattcattg ttggcgtact caatcaactt 900 aagttgagga tattctcgtt gcatgaactc ttgttcaaat ccccctttta ctacacctaa 960 tgagacgtta atggcccccg atagcagcgt atccaattta tcgcccaata acgtgcggtg 1020 tacgtagagt tgtgtatcga ttgtcagtaa aggttctgca aaatcgagat acgctaatct 1080 tgaagcagaa cggatcaaac cagcttgaac atcggatttg ccaagcttca ccgcttctag 1140 ggaatcattc caatccatca gttggaattc aatatcgaca tgattcgctt caccaaaagc 1200 caaccaaaaa tcaatcaata tgccagaagg ctgtccctgt tcatccaaat aagaataggg 1260 tttccatgct tttgagttgg caatagtcaa ggtttggcgc tctacagcct cactcattga 1320 tccgaataaa agcggccaag caatcatgag aagcagaaac agtttggtcg aaaagcgatg 1380 atccat 1386 <210> 4 <211> 461 <212> PRT
<213> Vibrio cholerae <400> 4 Met Asp His Arg Phe Ser Thr Lys Leu Phe Leu Leu Leu ntet Ile Ala Trp Pro Leu Leu Phe Gly Ser Met Ser Glu Ala val Glu Arg Gin Thr Leu Thr Ile Ala Asn Ser Lys Ala Trp Lys Pro Tyr Ser Tyr Leu Asp Glu Gin Gly Gin Pro Ser Gly Ile Leu Ile Asp Phe Trp Leu Ala Phe Gly Glu Ala Asn His Val Asp Ile Glu Phe Gin Leu Met Asp Trp Asn Asp Ser Leu Glu Ala Val Lys Leu Gly Lys Ser Asp val Gin Ala Gly 1 x Leu Ile Arg Ser Ala Ser Arg Leu Ala Tyr Leu Asp Phe Ala Glu Pro Leu Leu Thr Ile Asp Thr Gin Leu Tyr Val His Arg Thr Leu Leu Gly Asp Lys Leu Asp Thr Leu Leu Ser Gly Ala Ile Asn Val Ser Leu Gly val Val Lys Gly Gly Phe Glu Gln Glu Phe Met Gln Arg Glu Tyr Pro Gln Leu Lys Leu Ile Glu Tyr Ala Asn Asn Glu Leu Met Met Ser Ala Ala Lys Arg Arg Glu Leu Asp Gly Phe Val Ala Asp Thr Gln Val Ala Asn Phe Tyr Ile Val Val Ser Asn Gly Ala Lys Asp Phe Thr Pro Val Lys Phe Leu Tyr Ser Glu Glu Leu Arg Pro Ala Val Ala Lys Gly Asn Arg Asp Leu Leu Glu Gln Val Glu Gin Gly Phe Ala Gln Leu Ser Ser Asn Glu Lys Asn Arg Ile Leu Ser Arg Trp Val His Ile Glu Thr Ile Tyr Pro Arg Tyr Leu met Pro Ile Leu Ala Ser Gly Leu Leu Leu Ser Ile Val Ile Tyr Thr Leu Gln Leu Arg Arg Thr Val Arg Leu Arg Thr Gln Gln Leu Glu Glu Ala Asn Gln Lys Leu Ser Tyr Leu Ala Lys Thr Asp Ser Leu Thr Gly Ile Ala Asn Arg Arg Ser Phe Phe Glu His Leu Glu Ala Glu Gln Thr Arg Ser Gly Ser Leu Thr Leu met val Phe Asp Ile Asp Asp Phe Lys Thr Ile Asn Asp Arg Phe Gly His Gly Ala Gly Asp Asn Ala Ile Cys Phe val Val Gly Cys Val Arg Gln Ala Leu Ala Ser Asp Thr Tyr Phe Ala Arg Ile Gly Gly Glu Glu Phe Ala Ile Val Ala Arg Gly Lys Asn Ala Glu Glu Ser Gln Gln Leu Ala Glu Arg Ile Cys Gln Arg Val Ala Glu Lys Lys Trp Val Val Asn Ala Gln His Ser Leu Ser Leu Thr Ile Ser Leu Gly Cys Ala Phe Tyr Leu His Pro Ala Arg Pro Phe Ser Leu His Asp Ala Asp Ser Leu Met Tyr Glu Gly Lys Arg Asn Gly Lys Asn Gln Val Val Phe Arg Thr Trp Ser <210> 5 <211> 927 <212> DNA
<213> Vibrio cholerae <400> 5 atgatagaac ttaatagaat tgaagagctt tttgataacc aacagttctc cttgcacgaa 60 ctcgtgttga acgaactggg agtctatgtc ttcgtcaaaa atcgccgcgg cgagtatctc 120 tatgctaacc ctctgactct aaagttgttt gaagcggatg cacaatcgtt gtttggcaag 180 accgatcacg atttttttca tgatgatcaa ctcagtgata tcttggcggc cgatcaacag 240 gtgtttgaaa ctcgtctctc ggttatccat gaagaacgag ccatcgccaa atccaatggt 300 ttggttcgga tttatcgcgc agtcaaacac cctatcttgc accgagtgac aggcgaagtg 360 attgggctga ttggagtttc aaccgatatc accgatatcg tggaactgcg tgagcagcta 420 tatcagctcg ccaataccga ttctttaact cagctgtgta atcggcgtaa attgtgggcc 480 gattttcgcg ccgccttcgc tcgcgcaaaa cgtttaagac agccgttaag ttgcatctct 540 atcgatattg ataatttcaa actgatcaat gaccaatttg gtcacgataa aggtgatgaa 600 gtcctgtgtt ttctcgccaa actatttcag agcgtcatct ctgaccatca tttttgtggt 660 cgtgtgggag gtgaagagtt catcatcgtt ttggaaaata cgcacgtaga gacggctttt 720 catttggctg aacagatccg ccaacgtttt gcagagcatc cgttctttga acaaaacgag 780 cacatctacc tctgtgcggg ggtttccagc ttgcatcatg gtgatcatga cattgccgat 840 atttatcgac gctccgatca agcactgtat aaagccaagc gtaatggtcg taaccgttgc 900 tgtatctatc gccaatccac agaataa 927 <210> 6 <211> 308 <212> PRT
<213> Vibrio cholerae , <400> 6 Met Ile Glu Leu Asn Arg Ile Glu Glu Leu Phe Asp Asn Gln Gln Phe Ser Leu His Glu Leu Val Leu Asn Glu Leu Gly Val Tyr Val Phe Val Lys Asn Arg Arg Gly Glu Tyr Leu Tyr Ala Asn Pro Leu Thr Leu Lys Leu Phe Glu Ala Asp Ala Gln Ser Leu Phe Gly Lys Thr Asp His Asp Phe Phe His Asp Asp Gln Leu Ser Asp Ile Leu Ala Ala Asp Gln Gln Val Phe Glu Thr Arg Leu Ser Val Ile His Glu Glu Arg Ala Ile Ala Lys Ser Asn Gly Leu Val Arg Ile Tyr Arg Ala Val Lys His Pro Ile Leu His Arg Val Thr Gly Glu Val Ile Gly Leu Ile Gly Val Ser Thr Asp Ile Thr Asp Ile Val Glu Leu Arg Glu Gln Leu Tyr Gln Leu Ala Asn Thr Asp Ser Leu Thr Gln Leu Cys Asn Arg Arg Lys Leu Trp Ala Asp Phe Arg Ala Ala Phe Ala Arg Ala Lys Arg Leu Arg Gln Pro Leu Ser Cys Ile Ser Ile Asp Ile Asp Asn Phe Lys Leu Ile Asn Asp Gln Phe Gly His Asp Lys Gly AS Glu Val Leu Cys Phe Leu Ala Lys Leu Phe Gln Ser Val Ile Ser Asp His His Phe Cys Gly Arg Val Gly Gly Glu Glu Phe Ile Ile Val Leu Glu Asn Thr His Val Glu Thr Ala Phe His Leu Ala Glu Gln Ile Arg Gln Arg Phe Ala Glu His Pro Phe Phe Glu Gln Asn Glu His Ile Tyr Leu Cys Ala Gly Val Ser Ser Leu His His Gly Asp His Asp Ile Ala Asp Ile Tyr Arg Arg ser Asp Gin Ala Leu Tyr Lys Ala Lys Arg Asn Gly Arg Asn Arg Cys cys Ile Tyr Arg Gin Ser Thr Glu <210> 7 <211> 1374 <212> DNA
<213> vibrio cholerae <400> 7 tcaaaagcga tagagtgggt tttgcctacg cttagcggta tacatacgtt catcggccag 60 tttgaacatt tcatcaggtg tggcaaacga ctggtcatac aaagcatatc cgatacttac 120 acgaacatgg ataagcttgt cgtcataaac gatgggcgtt tcagaaatcc tttttaaaat 180 attgtcactg actttaagca cgtcttgttc acgatgaatt cgtggaatta acacgagaaa 240 ctcatccccc ccaatccgcg ccaccagatc ggaaacccgc aggctcgatt taattctttc 300 cgcacaagcc accagcactt tatcgcctgc gctatgtcca tgggaatcgt tgatagattt 360 aaaacggtca atatcaatgt tcaacaaagc aaagttacct tcgctatgag agcgcttagc 420 attttcaaag tagtgttcaa tggtatagat aaaatagcgc cgattcggca agtgggttaa 480 agggtcatgt agcgcacgct cctccgcgac ttgataaagg cgcatgataa cgccaaagcc 540 tgccatcaat accaataaca ccgagtatcc caacaagcgc actgcatttc gggtatacca 600 agataactgc tgtagtaaat cttgcttttc agcgaccgca attcgccaac ttccgtaagg 660 gaaatagaca ttctcttgtg caaaagcgtg ctcaaatact cgaggctctc caaaaaacac 720 gtccccctca ctgccacggc tgtctaaacc acgaatcgca acctgaaaat gctccccaaa 780 gctgtaaata ctggttgctg aaagcaatga atcccaatcc atcaccacac tcagtacccc 840 ccaataacgc gtatccttcg gtgggtcgta gaatatcggt tctcgaatca ccagcgcgcg 900 cccaccttga acgagatcga caggtccaga gacgaacgtc tgtttgattt cacgtgcttt 960 ttttattgac tgccactgct gaggaacggt gcggtaatcc aaaccgagta gtgcattggt 1020 ttgaggaagc ggatagctga aagcgaccac atcattaggg gcgataccta atgagcgtaa 1080 gtgatcgcta ttcctgatca ccgccgctga aagcggctcc cattgataga tattgaggtc 1140 gggatctagg gttaacaggg ttgttaaacc ttttacggta tagatatcac ccaaaatctc 1200 agcttctaat tgaaaacgta cgatggaaag atcttcttta gcttgttgac gtaaaccctc 1260 ttgtagatca cgtgtatggc taatatgaag ggattcaata accgcaatgc ccaaaaagag 1320 taaggcgaga aaataaattg agacatactt atatttgtgc gaggttaacc ccat 1374 <210> 8 I
<211> 457 <212> PRT
<213> vibrio cholerae <400> 8 met Gly Leu Thr Ser His Lys Tyr Lys Tyr Val Ser Ile Tyr Phe Leu Ala Leu Leu Phe Leu Gly Ile Ala Val Ile Glu Ser Leu His Ile Ser His Thr Arg Asp Leu Gin Glu Gly Leu Arg Gin Gin Ala Lys Glu Asp Leu Ser Ile Val Arg Phe Gin Leu Glu Ala Glu Ile Leu Gly Asp Ile Tyr Thr Val Lys Gly Leu Thr Thr Leu Leu Thr Leu Asp Pro Asp Leu Asn Ile Tyr Gin Trp Glu Pro Leu Ser Ala Ala Val Ile Arg Asn Ser Asp His Leu Arg Ser Leu Gly Ile Ala Pro Asn Asp Val Val Ala Phe Ser Tyr Pro Leu Pro Gin Thr Asn Ala Leu Leu Gly Leu Asp Tyr Arg Thr Val Pro Gin Gin Trp Gin Ser Ile Lys Lys Ala Arg Glu Ile Lys Gin Thr Phe Val Ser Gly Pro Val Asp Leu Val Gin Gly Gly Arg Ala Leu Val Ile Arg Glu Pro Ile Phe Tyr Asp Pro Pro Lys Asp Thr Arg Tyr Trp Gly Val Leu Ser val val met Asp Trp Asp Ser Leu Leu Ser Ala Thr Ser Ile Tyr Ser Phe Gly Glu His Phe Gin val Ala Ile Arg Gly Leu Asp Ser Arg Gly Ser Glu Gly Asp Val Phe Phe Gly Glu Pro Arg Val Phe Glu His Ala Phe Ala Gin Glu Asn Val Tyr Phe Pro Tyr Gly Ser Trp Arg Ile Ala Val Ala Glu Lys Gin Asp Leu Leu Gin Gin 1 , Leu Ser Trp Tyr Thr Arg Asn Ala val Arg Leu Leu Gly Tyr Ser Val Leu Leu val Leu Met Ala Gly Phe Gly Val Ile Met Arg Leu Tyr Gln Val Ala Glu Glu Arg Ala Leu His Asp Pro Leu Thr His Leu Pro Asn Arg Arg Tyr Phe Ile Tyr Thr Ile Glu His Tyr Phe Glu Asn Ala Lys Arg Ser His Ser Glu Gly Asn Phe Ala Leu Leu Asn Ile Asp Ile Asp Arg Phe Lys Ser Ile Asn Asp Ser His Gly His Ser Ala Gly Asp Lys Val Leu Val Ala Cys Ala Glu Arg Ile Lys Ser Ser Leu Arg Val Ser Asp Leu Val Ala Arg Ile Gly Gly Asp Glu Phe Leu val Leu Ile Pro Arg Ile His Arg Glu Gin Asp Val Leu Lys Val Ser Asp Asn Ile Leu Lys Arg Ile Ser Glu Thr Pro Ile Val Tyr Asp Asp Lys Leu Ile His Val Arg Val Ser Ile Gly Tyr Ala Leu Tyr Asp Gin Ser Phe Ala Thr Pro Asp Glu Met Phe Lys Leu Ala Asp Glu Arg Met Tyr Thr Ala Lys Arg Arg Gin Asn Pro Leu Tyr Arg Phe <210> 9 <211> 1374 <212> DNA
<213> Vibrio cholerae <400> 9 tcaaaagcga tagagtgggt tttgcctacg cttagcggta tacatacgtt catcggccag 60 tttgaacatt tcatcaggtg tggcaaacga ctggtcatac aaagcatatc cgatacttac 120 acgaacatgg ataagcttgt cgtcataaac gatgggcgtt tcagaaatcc tttttaaaat 180 attgtcactg actttaagca cgtcttgttc acgatgaatt cgtggaatta acacgagaaa 240 ctcatccccc ccaatccgcg ccaccagatc ggaaacccgc aggctcgatt taattctttc 300 cgcacaagcc accagcactt tatcgcctgc gctatgtcca tgggaatcgt tgatagattt 360 aaaacggtca atatcaatgt tcaacaaagc aaagttacct tcgctatgag agcgcttagc 420 attttcaaag tagtgttcaa tggtatagat aaaatagcgc cgattcggca agtgggttaa 480 agggtcatgt agcgcacgct cctccgcgac ttgataaagg cgcatgataa cgccaaagcc 540 tgccatcaat accaataaca ccgagtatcc caacaagcgc actgcatttc gggtatacca 600 agataactgc tgtagtaaat cttgcttttc agcgaccgca attcgccaac ttccgtaagg 660 gaaatagaca ttctcttgtg caaaagcgtg ctcaaatact cgaggctctc caaaaaacac 720 gtccccctca ctgccacggc tgtctaaacc acgaatcgca acctgaaaat gctccccaaa 780 gctgtaaata ctggttgctg aaagcaatga atcccaatcc atcaccacac tcagtacccc 840 ccaataacgc gtatccttcg gtgggtcgta gaatatcggt tctcgaatca ccagcgcgcg 900 cccaccttga acgagatcga caggtccaga gacgaacgtc tgtttgattt cacgtgcttt 960 ttttattgac tgccactgct gaggaacggt gcggtaatcc aaaccgagta gtgcattggt 1020 ttgaggaagc ggatagctga aagcgaccac atcattaggg gcgataccta atgagcgtaa 1080 gtgatcgcta ttcctgatca ccgccgctga aagcggctcc cattgataga tattgaggtc 1140 gggatctagg gttaacaggg ttgttaaacc ttttacggta tagatatcac ccaaaatctc 1200 agcttctaat tgaaaacgta cgatggaaag atcttcttta gcttgttgac gtaaaccctc 1260 ttgtagatca cgtgtatggc taatatgaag ggattcaata accgcaatgc ccaaaaagag 1320 taaggcgaga aaataaattg agacatactt atatttgtgc gaggttaacc ccat 1374 <210> 10 <211> 457 <212> PRT
<213> Vibrio cholerae <400> 10 Met Gly Leu Thr Ser His Lys Tyr Lys Tyr Val Ser Ile Tyr Phe Leu Ala Leu Leu Phe Leu Gly Ile Ala Val Ile Glu Ser Leu His Ile Ser His Thr Arg Asp Leu Gin Glu Gly Leu Arg Gin Gin Ala Lys Glu Asp Leu Ser Ile Val Arg Phe Gin Leu Glu Ala Glu Ile Leu Gly Asp Ile Tyr Thr Val Lys Gly Leu Thr Thr Leu Leu Thr Leu Asp Pro Asp Leu Asn Ile Tyr Gin Trp Glu Pro Leu Ser Ala Ala Val Ile Arg Asn Ser Asp His Leu Arg Ser Leu Gly Ile Ala Pro Asn Asp Val Val Ala Phe Ser Tyr Pro Leu Pro Gin Thr Asn Ala Leu Leu Gly Leu Asp Tyr Arg Thr Val Pro Gin Gin Trp Gin Ser Ile Lys Lys Ala Arg Glu Ile Lys Gin Thr Phe Val Ser Gly Pro Val Asp Leu Val Gin Gly Gly Arg Ala Leu Val Ile Arg Glu Pro Ile Phe Tyr AS Pro Pro Lys Asp Thr Arg Tyr Trp Gly Val Leu Ser val Val met Asp Trp Asp Ser Leu Leu Ser Ala Thr Ser Ile Tyr Ser Phe Gly Glu His Phe Gin Val Ala Ile Arg Gly Leu Asp Ser Arg Gly Ser Glu Gly Asp Val Phe Phe Gly Glu Pro Arg Val Phe Glu His Ala Phe Ala Gin Glu Asn val Tyr Phe Pro Tyr Gly Ser Trp Arg Ile Ala val Ala Glu Lys Gin Asp Leu Leu Gin Gin Leu Ser Trp Tyr Thr Arg Asn Ala Val Arg Leu Leu Gly Tyr Ser Val Leu Leu Val Leu Met Ala Gly Phe Gly Val Ile Met Arg Leu Tyr Gin val Ala Glu Glu Arg Ala Leu His Asp Pro Leu Thr His Leu Pro Asn Arg Arg Tyr Phe Ile Tyr Thr Ile Glu His Tyr Phe Glu Asn Ala Lys Arg Ser His Ser Glu Gly Asn Phe Ala Leu Leu Asn Ile Asp Ile Asp Arg Phe Lys Ser Ile Asn Asp Ser His Gly His Ser Ala Gly Asp Lys Val Leu Val Ala Cys Ala Glu Arg Ile Lys Ser Ser Leu Arg Val Ser Asp Leu Val Ala Arg Ile Gly Gly Asp Glu Phe Leu Val Leu Ile Pro Arg Ile His Arg Glu Gin Asp Val Leu Lys Val Ser Asp Asn Ile Leu Lys Arg Ile Ser Glu Thr Pro Ile Val Tyr Asp Asp Lys Leu Ile His val Arg Val Ser Ile Gly Tyr Ala Leu Tyr Asp Gin Ser Phe Ala Thr Pro Asp Glu Met Phe Lys Leu Ala Asp Gill Arg Met Tyr Thr Ala Lys Arg Arg Gin Asn Pro Leu Tyr Arg Phe <210> 11 <211> 1176 <212> DNA
<213> Vibrio cholerae <400> 11 atggatagct ttgctggcaa ccaattaaaa gagatgacag agatgcgttt tgctcgtaag 60 cagcatattg tcctgatcag ctctggtgtt gctaccgcta tttttcttgg gtttgccctt 120 tactactatt ttaaccatca acccctgtca tccggtttat tgttattaag cggtattgtc 180 accttattga atatgatttc gctgaatcgt caccgcgaat tacacactca agccgattta 240 attctgtcat taattctgct cacttatgcg ctggccttag tcagcaatgc tcagcatgaa 300 ttatcgcatc tcttatggtt atatccgctc atcaccactt tagtcatgat taaccctttt 360 cggttaggct tggtttacag tgcagcgata tgcttagcga tgaccgcctc tatccttttt 420 aatccggcac aaactggctc gtaccctatt gcacagacct attttttagt aagtctattt 480 acgctgacga ttatctgtaa taccgcttct ttctttttct caaaagcgat caattatatt 540 cataccctat accaagaagg tattgaagag ttggcttatc ttgatccgtt aacgggctta 600 gccaatcgtt ggagctttga aacttgggcc acagaaaagc tcaaagaaca acagagttcg 660 aataccatta ccgcgcttgt ttttctggat attgataatt tcaaacgcat taatgacagt 720 tacggccatg atgttggcga tcaggtgtta aaacattttg cacaccgtct acgcaataat 780 attcgtaata aagatcgagc caccaatcaa catgattatt ccattgctcg atttgctggt 840 gatgagtttg tgctcttgtt atatggtgtg cgaaatttgc gtgatctcga taatattctc 900 aaccgtatct gtaatctctt cgtcgaccgc tatcctgaga cggatatgct caacaacctc 960 acggtgagta taggggcagc tatttatccc aaagatgcga tcactctgcc ggaactaacc 1020 cgctgcgcag ataaagccat gtatgccgct aaacacggtg gaaaaaatca gtaccgctat 1080 taccatgatg ccgctttccc tccggctgta gaaaccgtat taggcagtca gcccgttgag 1140 , gctcciaacg taactccact gaaaaaagcg cactaa 1176 <210> 12 <211> 391 <212> PRT
<213> Vibrio cholerae <400> 12 Met Asp Ser Phe Ala Gly Asn Gln Leu Lys Glu Met Thr Glu Met Arg Phe Ala Arg Lys Gln His Ile Val Leu Ile Ser Ser Gly Val Ala Thr Ala Ile Phe Leu Gly Phe Ala Leu Tyr Tyr Tyr Phe Asn His Gln Pro Leu Ser Ser Gly Leu Leu Leu Leu Ser Gly Ile Val Thr Leu Leu Asn Met Ile Ser Leu Asn Arg His Arg Glu Leu His Thr Gln Ala AS Leu Ile Leu Ser Leu Ile Leu Leu Thr Tyr Ala Leu Ala Leu Val Ser Asn Ala Gln His Glu Leu Ser His Leu Leu Trp Leu Tyr Pro Leu Ile Thr Thr Leu Val Met Ile Asn Pro Phe Arg Leu Gly Leu Val Tyr Ser Ala Ala Ile Cys Leu Ala Met Thr Ala Ser Ile Leu Phe Asn Pro Ala Gln Thr Gly Ser Tyr Pro Ile Ala Gln Thr Tyr Phe Leu Val Ser Leu Phe Thr Leu Thr Ile Ile Cys Asn Thr Ala Ser Phe Phe Phe Ser Lys Ala Ile Asn Tyr Ile His Thr Leu Tyr Gln Glu Gly Ile Glu Glu Leu Ala Tyr Leu Asp Pro Leu Thr Gly Leu Ala Asn Arg Trp Ser Phe Glu Thr Trp Ala Thr Glu Lys Leu Lys Glu Gln Gln Ser Ser Asn Thr Ile Thr Ala Leu Val Phe Leu Asp Ile Asp Asn Phe Lys Arg Ile Asn Asp Ser Tyr Gly His Asp Val Gly Asp Gin Val Leu Lys His Phe Ala His Arg Leu Arg Asn Asn Ile Arg Asn Lys Asp Arg Ala Thr Asn Gin His Asp Tyr Ser Ile Ala Arg Phe Ala Gly Asp Glu Phe Val Leu Leu Leu Tyr Gly Val Arg Asn Leu Arg Asp Leu Asp Asn Ile Leu Asn Arg Ile Cys Asn Leu Phe Val Asp Arg Tyr Pro Glu Thr Asp Met Leu Asn Asn Leu Thr Val Ser Ile Gly Ala Ala Ile Tyr Pro Lys Asp Ala Ile Thr Leu Pro Glu Leu Thr Arg Cys Ala Asp Lys Ala Met Tyr Ala Ala Lys His Gly Gly Lys Asn Gin Tyr Arg Tyr Tyr His Asp Ala Ala Phe Pro Pro Ala Val Glu Thr Val Leu Gly Ser Gin Pro Val Glu Ala Pro Asn Val Thr Pro Leu Lys Lys Ala His <210> 13 <211> 447 <212> DNA
<213> Vibrio cholerae <400> 13 atgctagcgt tacctgcgga gtttgagcaa ttccattgga tggtcgatat ggttcagaat 60 gtcgatatgg gattgattgt gattaaccga gactacaacg tgcaagtgtg gaatgggttt 120 atgacccatc atagcggtaa gcaagctcat gatgttattg gtaaatctct gttcgagatt 180 tttccagaga tccctgtgga gtggtttaag ttaaaaacca aaccggtgta cgatctgggt 240 tgccgtagtt ttattacttg gcagcagcgc ccttatttgt tccattgccg taatgtgcgc 300 ccagtgactc agcaagccaa atttatgtat caaaacgtca cgcttaaccc aatgcgtaca 360 ccgacaggcg cgataaattc actcttctta tccattcaag atgcaacaag tgaagccctt 420 gtttctcaac aagcttcttc tcaataa 447 <210> 14 <211> 148 <212> PRT
<213> Vibrio cholerae <400> 14 Met Leu Ala Leu Pro Ala Glu Phe Glu Gln Phe His Trp Met Val Asp Met Val Gin Asn Val Asp Met Gly Leu Ile Val Ile Asn Arg Asp Tyr Asn Val Gin Val Trp Asn Gly Phe Met Thr His His Ser Gly Lys Gin Ala His Asp Val Ile Gly Lys Ser Leu Phe Glu Ile Phe Pro Glu Ile Pro Val Glu Trp Phe Lys Leu Lys Thr Lys Pro Val Tyr Asp Leu Gly Cys Arg Ser Phe Ile Thr Trp Gin Gin Arg Pro Tyr Leu Phe His Cys Arg Asn Val Arg Pro Val Thr Gin Gin Ala Lys Phe Met Tyr Gin Asn Val Thr Leu Asn Pro Met Arg Thr Pro Thr Gly Ala Ile Asn Ser Leu Phe Leu Ser Ile Gin Asp Ala Thr Ser Glu Ala Leu Val Ser Gin Gin Ala Ser Ser Gin <210> 15 <211> 1518 <212> DNA
<213> Vibrio cholerae <400> 15 ttagacaaaa tttcgcacaa cgtatcgatc tcgtccgtgt tctttcgcat gataaagtgc 60 catatccgcc tgatggaaca aagagagata agactccatc tttggagaaa tagcatacac 120 accaccaatg ctcaccgtta gatattggca gagtgcatca accggatttg caatcgcgag 180 ctgctcgatt ttgcttctca tctgttgtgc atactgttct gcatcaaatg cacagtccga 240 agctaaaaca acacaaaact cttctccccc aaagcgcgcc acgattttct cgccatggaa 300 ctccaccgat tggagcacat cagcaacgga acataaggct tcatcgccag ccaaatgacc 360 aaagctgtca ttgaaacgtt tgaaaaaatc gatatcgaca agaaacagca ccagataggc 420 ttgcggacga tcgctcaaat aacttttaag ctgcttttct aaatggcgac gattggaaat 480 gcgggttagt ggatcatgct cagactgcca acgtaacact tgttgactat cctccaattg 540 tccgacgatt cggttgatcg tagtggcaaa ttctttcatc tccgatgaga taaaagtact 600 cgcatccggc atttttccgc ccgatgtttt aaattgttgc aacacttgac tggcggtcgt 660 gatcggtttg atcaaggcaa tcaccaccca taaattgact aagtacatca ccagtgaaaa 720 gaacagcaaa gcaagaattt cttcggttcg aatgaaggga ggatgcttaa tgtgatggtt 780 aattttaaac aacacactgg aattaccgct gtaatcgagt tgcttgatgt atgaaacatc 840 cacttcgtct tgcggtaagg gcgcatcatt tttacaggtt aagacttcaa tatcgacacc 900 agtggcttgc tcaaccacat tcgcaaactg ggcgcggact tttttaataa agattaagaa 960 acctttgtta caccctttcc catcactgtc acagacacga gccgtggcag ctaaataggg 1020 ctcatcctcc accaccatat aacgaacgga agtcgagatt tcatccacac ttaaacgtgt 1080 cgcctgctgt aaaatacgtg aaaaatccgg caataagtgc tcatagctag agctctgccc 1140 cgttgctgcg tcatatttct tgccccaaac caaattgccc tcaggatcat agataaatac 1200 gccatcgagg aattgtgaac tgaaagcgtg ctctccaata ttgctttgtg tgaactcaag 1260 ggtgggtttt gcaatgaagt ctgccatttc atcccaagcg gcataatctg ccaaagaagc 1320 ccccatcgcc ttacgttcta acgacaacaa ggtttcaacc cgctgcaact cggcctgttg 1380 taactgcagc acttgcgcaa cttcacgatc atgtgaccag aaatatttaa aggtcagata 1440 aaacattaaa aagcctaaca ccaccgctaa cgcattgagt gtcgttagcc agcgtaggct 1500 aaagttattt aaattcat 1518 <210> 16 <211> 505 <212> PRT
<213> Vibrio cholerae <400> 16 Met Asn Leu Asn Asn Phe Ser Leu Arg Trp Leu Thr Thr Leu Asn Ala Leu Ala val Val Leu Gly Phe Leu Met Phe Tyr Leu Thr he Lys Tyr Phe Trp Ser His Asp Arg Glu val Ala Gin Val Leu Gin Leu Gin Gin Ala Glu Leu Gin Arg val Glu Thr Leu Leu Ser Leu Glu Arg Lys Ala met Gly Ala Ser Leu Ala Asp Tyr Ala Ala Trp Asp Glu met Ala Asp Phe Ile Ala Lys Pro Thr Leu Glu Phe Thr Gin Ser Asn Ile Gly Glu His Ala Phe Ser Ser Gin Phe Leu Asp Gly Val Phe Ile Tyr Asp Pro Glu Gly Asn Leu Val Trp Gly Lys Lys Tyr Asp Ala Ala Thr Gly Gin , Ser Ser Ser Tyr Glu His Leu Leu Pro Asp Phe Ser Arg Ile Leu Gin Gin Ala Thr Arg Leu Ser Val Asp Glu Ile Ser Thr Ser Val Arg Tyr Met val val Glu Asp Glu Pro Tyr Leu Ala Ala Thr Ala Arg Val Cys Asp Ser Asp Gly Lys Gly Cys Asn Lys Gly Phe Leu Ile Phe Ile Lys Lys val Arg Ala Gin Phe Ala Asn Val Val Glu Gin Ala Thr Gly Val Asp Ile Glu Val Leu Thr Cys Lys Asn Asp Ala Pro Leu Pro Gin Asp Glu val Asp Val Ser Tyr Ile Lys Gin Leu Asp Tyr Ser Gly Asn Ser Ser val Leu Phe Lys Ile Asn His His Ile Lys His Pro Pro Phe Ile Arg Thr Glu Glu Ile Leu Ala Leu Leu Phe Phe Ser Leu Val Met Tyr Leu val Asn Leu Trp Val Val Ile Ala Leu Ile Lys Pro Ile Thr Thr Ala Ser Gin val Leu Gin Gin Phe Lys Thr Ser Gly Gly Lys Met Pro Asp Ala Ser Thr Phe Ile Ser Ser Glu Met Lys Glu Phe Ala Thr Thr Ile Asn Arg Ile val Gly Gin Leu Glu Asp Ser Gin Gin Val Leu Arg Trp Gin Ser Glu His Asp Pro Leu Thr Arg Ile Ser Asn Arg Arg HiS

Leu Glu Lys Gin Leu Lys Ser Tyr Leu Ser Asp Arg Pro Gin Ala Tyr Leu Val Leu Phe Leu Val Asp Ile Asp Phe Phe Lys Arg Phe Asn Asp Ser Phe Gly His Leu Ala Gly Asp Glu Ala Leu Cys Ser val Ala Asp Val Leu Gin Ser Val Glu Phe His Gly Glu Lys Ile Val Ala Arg Phe Gly Gly Glu Glu Phe Cys Val Val Leu Ala Ser Asp Cys Ala Phe Asp Ala Glu Gin Tyr Ala Gin Gin Met Arg Ser Lys Ile Glu Gin Leu Ala Ile Ala Asn Pro Val Asp Ala Leu Cys Gin Tyr Leu Thr val Ser Ile Gly Gly Val Tyr Ala Ile Ser Pro Lys Met Glu Ser Tyr Leu Ser Leu Phe His Gin Ala Asp Met Ala Leu Tyr His Ala Lys Glu His Gly Arg Asp Arg Tyr Val Val Arg Asn Phe Val <210> 17 <211> 1740 <212> DNA
<213> Vibrio cholerae <400> 17 ttagtggttt ggttgataaa ttgaggtctg attgcggcca ttcgctttgg cttggtataa 60 agcccgatcc gctagctcaa ccatttgctc aggtacatcc tcaggccgag gaataagcgt 120 cactatgcct aagctgacgg taatcctatc ggcaacctta gaatgatcat gtggaatcgc 180 taatccacga actttctcat ggattcgctc tgcgaccagt attgctccgg actgtggtgt 240 attgggcagc aaaataccaa actcttctcc cccgtagcgg gcaacacaat cagaatggcg 300 attggcgact tgagtaaagg caatcgctat ctgtttgagc gtctcatcgc ccatcaaatg 360 gccataagcg tcgttgtaat ctttgaaata atcgacatca cacagaatga tgcttaatgg 420 tttgccttca cgcacatgca aatgccagag ggtatgcagt tgttcatcaa aacgacgacg 480 attggcaaca tgagtcaagc tatctaaaaa gcttaggcgt tccagctctt ggttggcggc 540 ttctaattgt tcagcggcga gatagcgctc cgacacatct cgcgccatga tcagcacgcc 600 attggtgccc gaagccggat ctcgaaaagg cgatttcaca acatcaaacc agataaactc 660 accatctgag cgttcaattc tgtcgatgta gcgcagagac ttaccttggt gcaggacttg 720 gctatccgta tcggaaagac gcgcatagat gtgctcgggg atcacatctt gcagccgttt 780 accaaccaga tctgacactt ccgcgatccc gagagcttcc acaaacggct ggttacaggc 840 ttggtagacc atgttttcat tgaagatacc aatcgaatcg gggctagatt ctaagatgtt 900 ttgtaaaatc gtatcgcgct gtgccaatgc cacttcggtg tcacggcgtt tttccatctc 960 . , ttctcttaat tgacgctgca tgttgtacca gtcggtcaca tcatgactga tgccaagtag 1020 cccaatattt tcaccttgcg gcgacatcaa tacccgttgg taggtttcta acagacagct 1080 gcgcccatca ggcgtcacag tccagcaacg ctgactcgtg cgccctttca taatgccttt 1140 aaaagtagcg ctgccctctt caatccggcc ttgccaaaac tgatcaaacg ctcggttggt 1200 tgcgattaag tggccttcgg tacttttaat aaaaatcagc tcggagaggg aatcaagtgc 1260 cgtgcgcgct atcgccagtg agtggcgctc ttgttgaatg tcatggctgg gacactcaaa 1320 accaatcaca ttcactagcc ataatttctt cggccaacga cgtaagagcg aagctgagat 1380 ctctagagtt tgggtcaaat tgcccggcac aggccaaagc agagggacgg aacgcttttg 1440 ctgtgcactg ctggcgagcg ctcgataaaa agcttgctga ctctcttcac tctgctcggc 1500 agaaaacaga tagtgacgtc ccaccaagcg gatccccagt aacaaatacg cggcaagatt 1560 ggcacgtaaa acgcgatcct ctcctaccaa gagcatccct gacggtgcat ggtgaagtaa 1620 ctgaatccac tgttgaggtt gaacatagcg ctgccatcct gaaaaaagcc ataacccacc 1680 accaagcaca agcccggcag cgaacaagaa acgtacaaat tcagagagaa attcaggcat 1740 <210> 18 <211> 579 <212> PRT
<213> Vibrio cholerae <400> 18 Met Pro Glu Phe Leu Ser Glu Phe Val Arg Phe Leu Phe Ala Ala Gly Leu val Leu Gly Gly Gly Leu Trp Leu Phe Ser Gly Trp Gin Arg Tyr val Gin Pro Gin Gin Trp Ile Gin Leu Leu His His Ala Pro Ser Gly Met Leu Leu Val Gly Glu Asp Arg Val Leu Arg Ala Asn Leu Ala Ala Tyr Leu Leu Leu Gly Ile Arg Leu val Gly Arg His Tyr Leu Phe Ser Ala Glu Gin Ser Glu Glu Ser Gin Gin Ala Phe Tyr Arg Ala Leu Ala Ser Ser Ala Gin Gin Lys Arg Ser Val Pro Leu Leu Trp Pro val Pro Gly Asn Leu Thr Gin Thr Leu Glu Ile Ser Ala Ser Leu Leu Arg Arg Trp Pro Lys Lys Leu Trp Leu val Asn val Ile Gly Phe Glu cys Pro . .
Ser His Asp Ile Gin Gin Glu Arg His Ser Leu Ala Ile Ala Arg Thr Ala Leu Asp Ser Leu Ser Glu Leu Ile Phe Ile Lys Ser Thr Glu Gly His Leu Ile Ala Thr Asn Arg Ala Phe AS Gin Phe Trp Gin Gly Arg Ile Glu Glu Gly Ser Ala Thr Phe Lys Gly Ile Met Lys Gly Arg Thr Ser Gin Arg Cys Trp Thr Val Thr Pro Asp Gly Arg Ser Cys Leu Leu Glu Thr Tyr Gin Arg Val Leu Met Ser Pro Gin Gly Glu Asn Ile Gly Leu Leu Gly Ile Ser His AS val Thr Asp Trp Tyr Asn met Gin Arg Gln Leu Arg Glu Glu Met Glu Lys Arg Arg Asp Thr Glu val Ala Leu Ala Gin Arg Asp Thr Ile Leu Gin Asn Ile Leu Glu Ser Ser Pro Asp Ser Ile Gly Ile Phe Asn Glu Asn Met Val Tyr Gin Ala Cys Asn Gin Pro Phe Val Glu Ala Leu Gly Ile Ala Glu Val Ser Asp Leu val Gly Lys Arg Leu Gin Asp val Ile Pro Glu His Ile Tyr Ala Arg Leu Ser Asp Thr Asp Ser Gin val Leu His Gin Gly Lys Ser Leu Arg Tyr Ile Asp Arg Ile Glu Arg Ser Asp Gly Glu Phe Ile Trp Phe Asp val val Lys Ser Pro Phe Arg Asp Pro Ala Ser Gly Thr Asn Gly Val Leu Ile Met Ala Arg Asp Val Ser Glu Arg Tyr Leu Ala Ala Glu Gin Leu Glu Ala Ala Asn Gin Glu Leu Glu Arg Leu Ser Phe Leu Asp Ser Leu Thr . .
His Val Ala Asn Arg Arg Arg Phe Asp Glu Gin Leu His Thr Leu Trp His Leu His Val Arg Glu Gly Lys Pro Leu Ser Ile Ile Leu Cys Asp Val Asp Tyr Phe Lys Asp Tyr Asn Asp Ala Tyr Gly His Leu Met Gly Asp Glu Thr Leu Lys Gin Ile Ala Ile Ala Phe Thr Gin Val Ala Asn Arg His Ser Asp Cys Val Ala Arg Tyr Gly Gly Glu Glu Phe Gly Ile Leu Leu Pro Asn Thr Pro Gin Ser Gly Ala Ile Leu Val Ala Glu Arg Ile His Glu Lys Val Arg Gly Leu Ala Ile Pro His Asp His Ser Lys Val Ala Asp Arg Ile Thr Val Ser Leu Gly Ile val Thr Leu Ile Pro Arg Pro Glu Asp val Pro Glu Gin Met Val Glu Leu Ala Asp Arg Ala Leu Tyr Gin Ala Lys Ala Asn Gly Arg Asn Gin Thr Ser Ile Tyr Gln Pro Asn His <210> 19 <211> 1584 <212> DNA
<213> vibrio cholerae <400> 19 ttacataaag tcgaacatcc tacctgaatt gaaggcataa ttcgattcta ccttgctgca 60 ttgctgcgca atcgatacac gatttcgacc tttcgattta ctgagataga gctgatcatc 120 aacactctgt aaaatttccg gctcactgta ctcacagtta atgctcgccc caatactgat 180 ggttaaggtt aatggtgtct cggcattgag catcacaggt tctgcttcga ccactttacg 240 gatccgctct agataagtat aaagcgccgt ttcatcagta acggatgaca agatggcaaa 300 ctcatcaccg ccgaaacggg caaaaatatc cgattcaacc aactcttttt tgaccacatc 360 aaccacatgc gttaaagcgt aatcccccgc taaatgccca tagctgtcgt tgatttgctt 420 aaagcggtcg atatcaaatg aaatcaaggt aaaggattgt ttttcatcta acattttgca 480 caaatgctga ctaaagaagc ggcggttata gatgttggtc aaactgtcat gctccaccag 540 ataacgcagc tctgcggtac gctcctcaat catatctgtc agccgttgtt tctcttccag 600 ttgcattcgc atgatgtagc taagcagcag agaaataata acgccaccca accctaagcc 660 cagtagcacc cactcttcac tatggttaat cggctgatgc agttcaaact ccagcaccca 720 atcacggttt ggcaacacca atttgcgctc tattttgggt tcatcatccg ctcgccacat 780 cgggctttga taaagaaccg gactgtcttc cgaatcaaat ccggtgtcaa tcacgcgcat 840 atcgagatct tgttccatga cgctgatttg gaccagtttc tcgaaatagg tggataggcg 900 caccaccccg accatcacac caagtaagct gcgatcatct tctgaagaaa aaacagggtg 960 atagaccaac atgccatctt tgacgatcga cttatcaatc ccatcttgta gcaggcgcac 1020 tttatccgaa acattcggcc gacgattaac gacaatatcc gccagtattc gtttgaaacg 1080 ttcacgctcc gagtaaaagc ctaacagttt acgattgtca taattgagtg gataaatatc 1140 cgataaaacg tatttcgctt ggtcatccgt accgaaaccg tatttgatct ctcccgtttt 1200 tggcaccgtg tacaaagtga actcaggaaa acgttgctgc attcgcgcgg taaaagtttc 1260 agcctgaggc ggctcaactt tcactaacca ttgtaaagca atcaggcttt gtgaaccttt 1320 aagagtctct tctgcgaaag tgtgaaaacg cacccagtca tcgcttgtgc ttgagcggaa 1380 aaagttggcg gcagagccga taaaatggat atcaccatcg acaaactgtt gcagtgccat 1440 agtttgccta tccgcaaggt tttccagcag agtacgatta tggcgcagct gtaatgagta 1500 tgcggtgtaa accacaaaca cagtcagaag cagagaaaac aacagtacca gcaagggcac 1560 aatcacgcgc acatgtttga gcat 1584 <210> 20 <211> 527 <212> PRT
<213> vibrio cholerae <400> 20 Met Leu Lys His Val Arg Val Ile Val Pro Leu Leu Val Leu Leu Phe Ser Leu Leu Leu Thr val Phe val Val Tyr Thr Ala Tyr Ser Leu Gin Leu Arg His Asn Arg Thr Leu Leu Glu Asn Leu Ala Asp Arg Gin Thr Met Ala Leu Gin Gin Phe Val Asp Gly Asp Ile His Phe Ile Gly Ser Ala Ala Asn Phe Phe Arg Ser Ser Thr Ser Asp Asp Trp val Arg Phe His Thr Phe Ala Glu Glu Thr Leu Lys Gly Ser Gin Ser Leu Ile Ala Leu Gin Trp Leu Val Lys Val Glu Pro Pro Gin Ala Glu Thr Phe Thr Ala Arg Met Gin Gin Arg Phe Pro Glu Phe Thr Leu Tyr Thr Val Pro Lys Thr Gly Glu Ile Lys Tyr Gly Phe Gly Thr Asp Asp Gin Ala Lys Tyr Val Leu Ser Asp Ile Tyr Pro Leu Asn Tyr Asp Asn Arg Lys Leu Leu Gly Phe Tyr Ser Glu Arg Glu Arg Phe Lys Arg Ile Leu Ala Asp Ile val Val Asn Arg Arg Pro Asn Val Ser Asp Lys val Arg Leu Leu Gin Asp Gly Ile Asp Lys Ser Ile Val Lys Asp Gly Met Leu Val Tyr His Pro val Phe Ser Ser GIL' Asp Asp Arg Ser Leu Leu Gly val met Val Gly Val Val Arg Leu Ser Thr Tyr Phe Glu Lys Leu Val Gin Ile Ser Val Met Glu Gin Asp Leu Asp Met Arg val Ile Asp Thr Gly Phe Asp Ser Glu Asp Ser Pro Val Leu Tyr Gin Ser Pro Met Trp Arg Ala Asp Asp Glu Pro Lys Ile Glu Arg Lys Leu Val Leu Pro Asn Arg Asp Trp val Leu Glu Phe Glu Leu His Gin Pro Ile Asn His Ser Glu Glu Trp val Leu Leu Gly Leu Gly Leu Gly Gly val Ile Ile Ser Leu Leu Leu Ser Tyr Ile Met Arg Met Gin Leu Glu Glu Lys Gin Arg Leu Thr Asp Met Ile Glu Glu Arg Thr Ala Glu Leu Arg Tyr Leu val Glu His Asp Ser Leu Thr Asn Ile Tyr Asn Arg Arg Phe Phe Ser Gin His Leu cys Lys Met Leu Asp Glu Lys Gin Ser Phe Thr Leu Ile Ser Phe Asp Ile Asp Arg Phe Lys Gin Ile Asn Asp Ser Tyr Gly His Leu Ala Gly Asp Tyr Ala Leu Thr His Val Val Asp Val val Lys Lys Glu Leu Val Glu Ser Asp Ile Phe Ala Arg Phe Gly Gly Asp Glu Phe Ala Ile Leu Ser Ser Val Thr Asp Glu Thr Ala Leu Tyr Thr Tyr Leu Glu Arg Ile Arg Lys Val Val Glu Ala Glu Pro Val met Leu Asn Ala Glu Thr Pro Leu Thr Leu Thr Ile Ser Ile Gly Ala Ser Ile Asn Cys Glu Tyr Ser Glu Pro Glu Ile Leu Gin Ser Val Asp Asp Gin Leu Tyr Leu Ser Lys Ser Lys Gly Arg Asn Arg Val Ser Ile Ala Gin Gin cys Ser Lys val Glu Ser Asn Tyr Ala Phe Asn Ser Gly Arg met Phe Asp Phe Met <210> 21 <211> 1332 <212> DNA
<213> vibrio cholerae <400> 21 tcagctcact aaactggtgt gatcgtgctt atcttggtgg gcgcaataca ccgtattgcc 60 ggattgatgt tttgcggtgt acatcgcctc atcagcaata cgcaataatt caggtacttg 120 ggtcgcttgc tctggatata aggcgacacc aatactcacc ccaatctcta agctctcttg 180 gttaagttga agcggctttt gtagtttttc tagcatctga taagccttat tgataacgcc 240 actgtgatcc tgcagcagat ctaggcatac cacaaattca tcccccccta agcgcccaca 300 aaaatccgat tctcgtatcg accctttgag ccgttgagcg atttcttgca agacaagatc 360 acctacttcg tgccctttgg tgtcattgat ttctttaaat ttatctaagt caaaaaacag 420 caaagccagc ttcatgtttg agcgcttcgc tttaattaac gcgtgactaa gctgctgttt 480 aaaggcacgg cggttcaaaa tacctgtcaa tgaatctctt tctgacaaga aacgtaattc 540 cgctttttga cgctctaatt tggcggtttt tcttgccact tccgcttgta actcatcttt 600 ggtaacggtt gtgctttgca gcgaagcctt catttgattg aaaaactgag ttaattgaac 660 aaactcttgt tcattatttt gagtggaaat tcggctggcg agatcccctt tcgccatttg 720 ttcaatccct tcttggagag ttttacatcc atgtcggaag cggcgtaaca ccaccagcgc 780 gataccacag acaaccgatg agaagagcag taagtgcgcc atggtggtta acaataaata 840 gcgttgatta ttaatgctct cttccatgac ttgacgctga aaataggcca actcctcatt 900 catgttttgc accaaaatat tgtatcgaga gtgaagtagc tcataagttc cgatgccatc 960 gaccaactta gtaatgcccg attcttcggc catgtagcgc tcttgttcta atagcccggc 1020 taaactgtta ttcattcttt ggatgccggc taagtgttgc ccaaagaccg tttccatctc 1080 gagctgccca gccaaaacct gctgcgcacg ataaacctgc tctaagctat gagcatcgtt 1140 gtattgcaga aagacccaga gctggctacg caacatggca atgctgtttt ggatttccaa 1200 aatcgtatcc agctcagcat tggtttgctg ctgccgctga tctaagttca gtaatgagaa 1260 agcaataaaa ccaactaaca gcagtgatgc aataaacagt aacgtcattt tgcggtttaa 1320 tgagttgatc aa 1332 <210> 22 <211> 443 <212> PRT
<213> Vibrio cholerae <400> 22 Met Ile Asn Ser Leu Asn Arg Lys Met Thr Leu Leu Phe Ile Ala Ser Leu Leu Leu Val Gly Phe Ile Ala Phe Ser Leu Leu Asn Leu Asp Gin Arg Gin Gin Gin Thr Asn Ala Glu Leu Asp Thr Ile Leu Glu Ile Gin , Asn Ser Ile Ala Met Leu Arg Ser Gin Leu Trp Val Phe Leu Gin Tyr Asn Asp Ala His Ser Leu Glu Gin Val Tyr Arg Ala Gin Gin Val Leu Ala Gly Gin Leu Glu Met Glu Thr Val Phe Gly Gin His Leu Ala Gly Ile Gin Arg Met Asn Asn Ser Leu Ala Gly Leu Leu Glu Gin Glu Arg Tyr Met Ala Glu Glu Ser Gly Ile Thr Lys Leu Val Asp Gly Ile Gly Thr Tyr Glu Leu Leu His Ser Arg Tyr Asn Ile Leu Val Gin Asn Met Asn Glu Glu Leu' Ala Tyr Phe Gin Arg Gin val met Glu Glu Ser Ile Asn Asn Gin Arg Tyr Leu Leu Leu Thr Thr met Ala His Leu Leu Leu Phe Ser Ser Val Val Cys Gly Ile Ala Leu Val Val Leu Arg Arg Phe Arg His Gly Cys Lys Thr Leu Gin Glu Gly Ile Glu Gin Met Ala Lys Gly Asp Leu Ala Ser Arg Ile Ser Thr Gin Asn Asn Glu Gin Glu Phe Val Gin Leu Thr Gin Phe Phe Asn Gin Met Lys Ala Ser Leu Gin Ser Thr Thr Val Thr Lys Asp Glu Leu Gin Ala Glu val Ala Arg Lys Thr Ala Lys Leu Glu Arg Gin Lys Ala Glu Leu Arg Phe Leu Ser Glu Arg Asp Ser Leu Thr Gly Ile Leu Asn Arg Arg Ala Phe Lys Gin Gin Leu Ser His Ala Leu Ile Lys Ala Lys Arg Ser Asn Met Lys Leu Ala Leu Leu Phe Phe Asp Leu Asp Lys Phe Lys Glu Ile Asn Asp Thr Lys Gly His Glu Val Gly Asp Leu Val Leu Gin Glu Ile Ala Gin Arg Leu Lys Gly Ser Ile Arg Glu Ser Asp Phe Cys Gly Arg Leu Gly Gly Asp Glu Phe Val val Cys Leu Asp Leu Leu Gin AS His Ser Gly val Ile Asn Lys Ala Tyr Gin Met Leu Glu Lys Leu Gin Lys Pro Leu Gin Leu Asn Gin Glu Ser Leu Glu Ile Gly Val Ser Ile Gly Val Ala Leu Tyr Pro Glu Gin Ala Thr Gin Val Pro Glu Leu Leu Arg Ile Ala Asp Glu Ala Met Tyr Thr Ala Lys His Gin Ser Gly Asn Thr Val Tyr Cys Ala His Gin Asp Lys His Asp His Thr Ser Leu val Ser <210> 23 <211> 2022 <212> DNA
<213> Vibrio cholerae <400> 23 atgctactta acgctttttc acgccgagtc ttcctttggc taggttggct attgatttcc 60 accagcagtt tagccgctac atctacgacg tataaggtcg ccaccgaagc ggatgacgtg 120 gtgactcgtg tgctttttga ttcgattgct caccacttca accttgaaat tgaatacgtc 180 aactacccca gttttaacga tattctggtg gcgatagaga ctggcaacgc cgattttgct 240 gccaacatta cttacactga tttgcgtgct caacgttttg atttttcaag accaaccaac 300 atcgagtaca cctatctcta cagttatggt ggcctacgtt tacccgagtt gcgcctcgtg 360 ggtatcccga aaggaaccac ctacgggacc ctactaaaag aacactatcc ctatatccag 420 caagttgagt atgaagggca tttagaagcg ctcactttgc tggaaagtgg ccgagtagac 480 ggagtggttg atgcgatcaa tcagctcaaa cctatgctac tgaaagggct tgatgtacaa 540 ctccttaacg accaattacc gattcagcct gtttctattg tgacgcctaa aggcaaacac 600 tcagcgctat tgggcaagat tgaaaaatac gcgcattcgg ctcacgtaca acgtttattg 660 cgtgaatcga tccaaaagta tcaattggac atccgtaagc aagctctgcg tcaatccgtg 720 gttgagagcg gactcaacgt gcagcgtgta ttgcgtgtta agctagagaa caacccgcaa 780 tatgcacttt atcagccaga cggttcggtt cgtgggatca gtgctgatgt tgtgtttcag 840 gcctgtgaga tgctactgct gaaatgcgaa ttggtcagta atggtcaaga aacatgggag 900 agcatgtttg atgatttaca ggataaaagc atcgatattt tggctcctat aacggtttct 960 cagcagcgta aaaacctcgc ttacttcagt gaaagctact accacccaca agcgattttg 1020 gtcaaacgtg aacactataa agacgatgtg tatagcaatg tgtctgagtt ggtggctgaa 1080 cgtattggcg tcatcaaaga cgattttttt gaagagctgt tacagcagat gctgccgaac 1140 aagatcttgt tcagctacgc aagtcaggaa gagaaagttc aagccttact gaataaagag 1200 gtggactaca tagtgctcaa tagagccaat tttaatctct tgcttcgcga gtcaacggag 1260 atgttaccga ttgtagaaga caccatgatt ggcagtttct accaatatga cattgcgata 1320 ggttttgcta aaaatccact tggtgcaact ctggcacctc ttttctctcg ggcaattaaa 1380 atgctcaata ccgaacagat catacatacc tatgattatc agccaaattg gcgagccaca 1440 ttacttgcgg aaaagaaata tcagcgcagt actcaatggc tttttgccat ggctttcatc 1500 gttttgttta tggtggcgtt ttacctccat ggcatatcac ataccgataa ccttactaag 1560 ttgcgcaatc gtcgcgcttt gtataaccga taccgccgcg ggttatcgcc tcgcctaagc 1620 ttggtttatc ttgacgtgaa tacgtttaaa tcaatcaacg atcagtatgg acatgaagtg 1680 ggtgacaaag tccttaagca gttggctcag cgcatcgaag cggtatggcg tgggcgcagc 1740 tatcggattg gtggggatga atttatttta atcggtgaat gttctgctaa gcggcttgaa 1800 catgtggttg cgcaatgtga acgttttatg tttgtggatg cagagcgcga tgtcagtttt 1860 gaagtgagtg tggcgattgg tattgctaag aatcgtgagc ggaccgaatc actcaatgag 1920 gtgatgcacc aagcggatat tgcgatgtat cgcgctaagg cggaatcgac gcaatcgcca 1980 tttcaggctg ccagcaaggt aaaaggatta cacatcgttt aa 2022 <210> 24 <211> 673 <212> PRT
<213> vibrio cholerae <400> 24 met Leu Leu Asn Ala Phe Ser Arg Arg Val Phe Leu Trp Leu Gly Trp Leu Leu Ile Ser Thr Ser Ser Leu Ala Ala Thr Ser Thr Thr Tyr Lys Val Ala Thr Glu Ala Asp Asp Val Val Thr Arg Val Leu Phe Asp Ser Ile Ala His His Phe Asn Leu Glu Ile Glu Tyr Val Asn Tyr Pro Ser Phe Asn Asp Ile Leu Val Ala Ile Glu Thr Gly Asn Ala Asp Phe Ala Ala Asn Ile Thr Tyr Thr Asp Leu Arg Ala Gin Arg Phe Asp Phe Ser Arg Pro Thr Asn Ile Glu Tyr Thr Tyr Leu Tyr Ser Tyr Gly Gly Leu Arg Leu Pro Glu Leu Arg Leu Val Gly Ile Pro Lys Gly Thr Thr Tyr Gly Thr Leu Leu Lys Glu His Tyr Pro Tyr Ile Gin Gin val Glu Tyr Glu Gly His Leu Glu Ala Leu Thr Leu Leu Glu Ser Gly Arg Val Asp Gly Val Val Asp Ala Ile Asn Gin Leu Lys Pro Met Leu Leu Lys Gly Leu Asp Val Gin Leu Leu Asn Asp Gin Leu Pro Ile Gin Pro Val Ser ' , Ile val Thr Pro Lys Gly Lys His Ser Ala Leu Leu Gly Lys Ile Glu Lys Tyr Ala His Ser Ala His val Gin Arg Leu Leu Arg Glu Ser Ile Gin Lys Tyr Gin Leu Asp Ile Arg Lys Gin Ala Leu Arg Gin Ser Val val Glu Ser Gly Leu Asn Val Gin Arg val Leu Arg Val Lys Leu Glu Asn Asn Pro Gin Tyr Ala Leu Tyr Gin Pro Asp Gly Ser Val Arg Gly Ile Ser Ala Asp val val Phe Gin Ala Cys Glu Met Leu Leu Leu Lys cys Glu Leu Val Ser Asn Gly Gin Glu Thr Trp Glu Ser Met Phe Asp Asp Leu Gin Asp Lys Ser Ile Asp Ile Leu Ala Pro Ile Thr Val Ser Gin Gin Arg Lys Asn Leu Ala Tyr Phe Ser Glu Ser Tyr Tyr His Pro Gin Ala Ile Leu Val Lys Arg Glu His Tyr Lys Asp Asp val Tyr Ser Asn val Ser Glu Leu Val Ala Glu Arg Ile Gly val Ile Lys Asp Asp Phe Phe Glu Glu Leu Leu Gin Gin Met Leu Pro Asn Lys Ile Leu Phe Ser Tyr Ala Ser Gin Glu Glu Lys Val Gin Ala Leu Leu Asn Lys Glu val Asp Tyr Ile Val Leu Asn Arg Ala Asn Phe Asn Leu Leu Leu Arg Glu Ser Thr Glu Met Leu Pro Ile Val Glu Asp Thr Met Ile Gly Ser Phe Tyr Gin Tyr Asp Ile Ala Ile Gly Phe Ala Lys Asn Pro Leu Gly Ala Thr Leu Ala Pro Leu Phe Ser Arg Ala Ile Lys Met Leu Asn Thr , , . , Glu Gin Ile Ile His Thr Tyr Asp Tyr Gin Pro Asn Trp Arg Ala Thr Leu Leu Ala Glu Lys Lys Tyr Gin Arg Ser Thr Gin Trp Leu Phe Ala Met Ala Phe Ile Val Leu Phe Met Val Ala Phe Tyr Leu His Gly Ile ser His Thr Asp Asn Leu Thr Lys Leu Arg Asn Arg Arg Ala Leu Tyr Asn Arg Tyr Arg Arg Gly Leu Ser Pro Arg Leu Ser Leu Val Tyr Leu Asp Val Asn Thr Phe Lys Ser Ile Asn Asp Gin Tyr Gly His Glu val Gly Asp Lys Val Leu Lys Gin Leu Ala Gin Arg Ile Glu Ala Val Trp Arg Gly Arg Ser Tyr Arg Ile Gly Gly Asp Glu Phe Ile Leu Ile Gly Glu Cys Ser Ala Lys Arg Leu Glu His val Val Ala Gin Cys Glu Arg Phe met Phe Val Asp Ala Glu Arg Asp Val Ser Phe Glu Val Ser val Ala Ile Gly Ile Ala Lys Asn Arg Glu Arg Thr Glu Ser Leu Asn Glu val Met His Gin Ala Asp Ile Ala Met Tyr Arg Ala Lys Ala Glu Ser Thr Gin Ser Pro Phe Gin Ala Ala Ser Lys val Lys Gly Leu His Ile Val <210> 25 <211> 1293 <212> DNA
<213> Vibrio cholerae <400> 25 ctatctgaac tgatcctgct tgagttcttt cgcactggga agaggcagga tctcttcccc 60 cattcgataa atatgatagc catgtttgcc tctgtatttg acccagtaca tggctttatc 120 ggcttgtagc agcagttttt ctaagtcaat gtgcagactg ttcatatgac taatcccgat 180 actgcaaccc acttgcgcac tctgctgacc caatccaatc ggctcagagg aggattcgat 240 caactgagcc gcaaaccgct cgatagattc ggcaacaaat tcatccagcg gaatgtaaat 300 agcaaactca tcaccaccga gccgtccgac cacaaaatca gaaaaatgtg tttgcgccaa 360 ggcataaaaa cgtttggcga tttcacgtaa tacctcatcg ccagccgcat gccccaaggt 420 atcattcacc tgcttaaaac catccaaatc aatcaatagc agcaccatag tggtgctagc 480 acgctgtttg cggagcacga acttctcaca acctaaacgg ttttttagtc ccgtcagcgt 540 gtcttgttcg gcaatggtgc gatagtagct ttcccaacgt tcaatctgtt gacgcagctc 600 gcgttcacgc agtaaagctt gatgggaagc gtcgataaat tcattgatgc ttttggccac 660 caaaccaatc tcgttgtggt gatcttctgc ggctaccgcc actttgcgat catgatctgg 720 ccgcacttcg gataacgcct gtgaaagatc cgtcaggggt ttaccgacca agcggcgaac 780 gatccagata agcgcaataa aagtcacgag aaactggatc aaaaccacgg ctatctgatc 840 aagaatctga ttaatggctt gctgacgaat cacctgatga tcctcatgaa tcatcagata 900 gccaatcaaa ttaccatcta cgggagaatc taatcggtag cggttcgcat cactccaata 960 attctgctct ttgtaggttg aggggatggt ggtgcgctca aagacaatgc catccacgct 1020 ggctaactta accgcattga tctcttgatg aagcagcaac gcatccatca cctcggaggc 1080 aatatcgtaa ttattcacat acagtgcaat ggccgccgag ttactcaagg agagcgcaag 1140 cttctcttcc agctcttgtt tttgctgctc aacactctgt atgccgcgcg gaataatgat 1200 ggccaaaatg atcagcaaat acccaagtgc acacagtgaa atcatcttca gcaagcgatt 1260 aaccagtggc gaagttcgcg tttgatcagt cat 1293 <210> 26 <211> 430 <212> PRT
<213> Vibrio cholerae <400> 26 met Thr Asp Gin Thr Arg Thr Ser Pro Leu val Asn Arg Leu Leu Lys Met Ile Ser Leu Cys Ala Leu Gly Tyr Leu Leu Ile Ile Leu Ala Ile Ile Ile Pro Arg Gly Ile Gin Ser Val Glu Gin Gin Lys Gin Glu Leu Glu Glu Lys Leu Ala Leu Ser Leu Ser Asn Ser Ala Ala Ile Ala Leu Tyr val Asn Asn Tyr Asp Ile Ala Ser Glu val Met Asp Ala Leu Leu Leu His Gin Glu Ile Asn Ala Val Lys Leu Ala Ser val Asp Gly Ile , val Phe Glu Arg Thr Thr Ile Pro Ser Thr Tyr Lys Glu Gin Asn Tyr Trp Ser Asp Ala Asn Arg Tyr Arg Leu Asp Ser Pro Val Asp Gly Asn Leu Ile Gly Tyr Leu Met Ile His Glu Asp His Gin Val Ile Arg Gin Gin Ala Ile Asn Gin Ile Leu Asp Gin Ile Ala Val Val Leu Ile Gin Phe Leu val Thr Phe Ile Ala Leu Ile Trp Ile Val Arg Arg Leu Val Gly Lys Pro Leu Thr Asp Leu Ser Gin Ala Leu Ser Glu Val Arg Pro Asp His Asp Arg Lys Val Ala Val Ala Ala Glu Asp His His Asn Glu Ile Gly Leu Val Ala Lys Ser Ile Asn Glu Phe Ile Asp Ala Ser His Gin Ala Leu Leu Arg Glu Arg Glu Leu Arg Gin Gin Ile Glu Arg Trp Glu Ser Tyr Tyr Arg Thr Ile Ala Glu Gin Asp Thr Leu Thr Gly Leu Lys Asn Arg Leu Gly Cys Glu Lys Phe Val Leu Arg Lys Gin Arg Ala Ser Thr Thr met val Leu Leu Leu Ile Asp Leu Asp Gly Phe Lys Gin Val Asn Asp Thr Leu Gly His Ala Ala Gly Asp Glu Val Leu Arg Glu Ile Ala Lys Arg Phe Tyr Ala Leu Ala Gin Thr His Phe Ser Asp Phe Val Val Gly Arg Leu Gly Gly Asp Glu Phe Ala Ile Tyr Ile Pro Leu Asp Glu Phe Val Ala Glu Ser Ile Glu Arg Phe Ala Ala Gin Leu Ile Glu Ser Ser Ser Glu Pro Ile Gly Leu Gly Gin Gin Ser Ala Gin Val Gly Cys Ser Ile Gly Ile Ser His Met Asn Ser Leu His Ile Asp Leu Glu Lys Leu Leu Leu Gin Ala Asp Lys Ala Met Tyr Trp Val Lys Tyr Arg Gly Lys His Gly Tyr His Ile Tyr Arg Met Gly Glu Glu Ile Leu Pro Leu Pro Ser Ala Lys Glu Leu Lys Gin Asp Gin Phe Arg <210> 27 <211> 1003 <212> DNA
<213> Vibrio cholerae <400> 27 agcgcatacg ctcaagtagg gcttgctcac gttgctccgc taagagtaag cgttcagaaa 60 gtgaagacat ctcgcgcagt aaaggcgcca ttttcagctt gagctgttct agctccgtct 120 gttctttgag cgcggtctgg ctacgagcca ctaaactgct cagctcgcca ttcatctctt 180 ggcggtgtgc catgtaactc tggctttgct caagattttg agtcgcgctt tttaggttat 240 tgccaatcga gagattcact tgctcgagaa aggcttcggc tgctttgcgc tcagcatggc 300 ttccatcgac gactaaacgc agtacttcaa gggtgagctc aagcagggta tgggtattga 360 cgccaagcag aagcttggtt cggatatcgg tcagttgatc acccgattca ccattgaaat 420 ccaactcagt aatcaagtgt tgtaaatcaa cggcaagtcg atgcagcagt tctcgatccg 480 cttgttgagt aagctcattg agcgccaaat tgggattggc acattgaatt ttgaccgcgc 540 gttcataaat ttccagcaaa cgcaaagctt gctgagtttt ttccagcggc tgtgcggcgc 600 taaaactcag cagatctcga agatcgcgtt tgatcttggc gggtaagccg gggacgcgca 660 gtagcgtttC accactgtgc tgtagctggc tatccagatg actcgtttgt ttgtccatgg 720 ccaatgactg ttgtttcaac atgcgttcca gtacggctaa tttcgggatc agcgtactga 780 tgtctttttg ttgttctaat gcaaaacaga gttcttctaa actttggttt agtcgagagc 840 tactgccgcg gcaagtcgta gccaaggaag tgaccattcg tttaagaact tgctgctctc 900 ggttaaattt gaacgaagta tccctttgtg tcaaacgtac ttgttctaac tgagatttca 960 gtttttgaag ctctgcttgg atatcttgtt ctagaacgcc cat 1003 <210> 28 <211> 521 <212> PRT
<213> Vibrio cholerae <400> 28 met Gly Val Leu Glu Gin Asp Ile Gin Ala Glu Leu Gin Lys Leu Lys Ser Gin Leu Glu Gin val Arg Leu Thr Gin Arg Asp Thr Ser Phe Lys Phe Asn Arg Glu Gin Gin val Leu Lys Arg met Val Thr Ser Leu Ala Thr Thr Cys Arg Gly Ser Ser Ser Arg Leu Asn Gin Ser Leu Glu Glu Leu Cys Phe Ala Leu Glu Gin Gin Lys Asp Ile Ser Thr Leu Ile Pro Lys Leu Ala Val Leu Glu Arg met Leu Lys Gin Gin Ser Leu Ala met Asp Lys Gin Thr Ser His Leu Asp Ser Gin Leu Gin His Ser Gly Glu Thr Leu Leu Arg Val Pro Gly Leu Pro Ala Lys Ile Lys Arg Asp Leu Arg Asp Leu Leu Ser Phe Ser Ala Ala Gin Pro Leu Glu Lys Thr Gin Gin Ala Leu Arg Leu Leu Glu Ile Tyr Glu Arg Ala Val Lys Ile Gin cys Ala Asn Pro Asn Leu Ala Leu Asn Glu Leu Thr Gin Gin Ala Asp Arg Glu Leu Leu His Arg Leu Ala Val Asp Leu Gin His Leu Ile Thr Glu Leu Asp Phe Asn Gly Glu Ser Gly Asp Gin Leu Thr Asp Ile Arg Thr Lys Leu Leu Leu Gly Val Asn Thr His Thr Leu Leu Glu Leu Thr Leu Glu Val Leu Arg Leu Val val Asp Gly Ser His Ala Glu Arg Lys Ala Ala Glu Ala Phe Leu Glu Gin Val Asn Leu Ser Ile Gly Asn Asn Leu Lys Ser Ala Thr Gin Asn Leu Glu Gin Ser Gin Ser Tyr Met Ala His Arg Gin Glu met Asn Gly Glu Leu Ser Ser Leu Val Ala Arg Ser Gin Thr Ala Leu Lys Glu Gin Thr Glu Leu Glu Gin Leu Lys Leu Lys Met Ala Pro Leu Leu Arg Glu Met Ser Ser Leu Ser Glu Arg Leu Leu Leu Ala Glu Gin Arg Glu Gin Ala Leu Leu Glu Arg Met Arg Tyr Ser Lys Asp Gln Met Glu Ala Leu Ser Asp Leu Ala Gin Asp Tyr Arg Arg Arg Leu Glu Asp Gin Ala Leu Arg Ala Gin Leu Asp Pro Leu Thr Lys Val Tyr Asn Arg Ser Ser Phe Thr Glu Arg Leu Glu His Glu Tyr Arg Arg Trp Ile Arg Thr Gin His Asn Leu Arg Val Val Leu Phe Asp Ile Asp Lys Phe Lys Ser Ile Asn Asp Ser Phe Gly Tyr Thr Ala Gly Asp Lys Ala Leu Ser Ile Ile Ala Arg Thr Ile Lys Lys Giu Leu Arg Asp Ser Asp Thr Val Ala Arg Phe Ser Gly GIL, Glu Phe Ile Leu Leu Leu Pro Glu Arg Ser Asp Asn Glu Ser Tyr Gin Ile Ile His Gin Ile Gin Leu Asn Val Ser Lys Leu Pro Phe Lys Phe Arg Asp Lys Ser Leu Thr Ile Thr Leu Ser Ala Ala Ser Ile Arg Phe Met Asp Ser Asp Thr Pro _ Glu Thr Val Leu Asp Arg Leu Asn Leu Thr Leu Ser Glu Ala Lys His Ile Gly Pro Ser Gin Leu Ala Trp Lys <210> 29 <211> 563 <212> DNA
<213> Vibrio cholerae <400> 29 atagcaaaga tcagatggaa gccctgtctg atttggcaca agattatcgt cgccgccttg aagatcaagc attgcgcgca caactcgatc ctctgaccaa agtgtacaac cgcagcagct 120 ttactgagcg acttgaacat gagtatcgcc gctggatccg tacgcaacac aatttgcggg 180 tagtgctgtt tgatattgat aaattcaaat cgatcaacga cagctttggc tacaccgcag 240 gcgataaggc cttaagtatc attgctcgca ccatcaaaaa agaattacga gacagtgaca 300 ccgtggctcg cttctctggt gaagagttca ttctgttact gcctgaacgc tccgataatg 360 agagttacca gattattcac cagatccagc tcaacgtgtc gaaactaccg ttcaagttcc 420 gcgataagag cctaaccatc acgctgtctg cggcgagtat ccgcttcatg gattcagata 480 cccccgaaac ggttcttgat cgtttaaatc tgacgctaag tgaagccaaa catatcggtc 540 caagtcagtt agtttggaaa taa 563 <210> 30 <211> 434 <212> PRT
<213> Vibrio cholerae <400> 30 Met Leu Lys Gin Gin Ser Leu Ala Met Asp Lys Gin Thr Ser His Leu Asp Ser Gin Leu Gin His Ser Gly Glu Thr Leu Leu Arg Val Pro Gly Leu Pro Ala Lys Ile Lys Arg Asp Leu Arg Asp Leu Leu Ser Phe Ser Ala Ala Gin Pro Leu Glu Lys Thr Gin Gin Ala Leu Arg Leu Leu Glu Ile Tyr Glu Arg Ala Val Lys Ile Gin Cys Ala Asn Pro Asn Leu Ala Leu Asn Glu Leu Thr Gin Gin Ala Asp Arg Glu Leu Leu His Arg Leu Ala val Asp Leu Gin His Leu Ile Thr Glu Leu Asp Phe Asn Gly Glu Ser Gly Asp Gin Leu Thr Asp Ile Arg Thr Lys Leu Leu Leu Gly Val Asn Thr His Thr Leu Leu Glu Leu Thr Leu Glu val Leu Arg Leu Val Val Asp Gly Ser His Ala Glu Arg Lys Ala Ala Glu Ala Phe Leu Glu Gin Val Asn Leu Ser Ile Gly Asn Asn Leu Lys Ser Ala Thr Gin Asn , , Leu Glu Gin Ser Gin Ser Tyr Met Ala His Arg Gin Glu met Asn Gly Glu Leu Ser Ser Leu Val Ala Arg Ser Gin Thr Ala Leu Lys Glu Gln Thr Glu Leu Glu Gin Leu Lys met Lys Met Ala Pro Leu Leu Arg Glu Met Ser Ser Leu Ser Glu Arg Leu Leu Leu Ala Glu Gin Arg Glu Gin Ala Leu Leu Glu Arg Met Arg Tyr Ser Lys Asp Gin Met Glu Ala Leu Ser Asp Leu Ala Gin Asp Tyr Arg Arg Arg Leu Glu Asp Gin Ala Leu Arg Ala Gin Leu Asp Pro Leu Thr Lys Val Tyr Asn Arg Ser Ser Phe Thr Glu Arg Leu Glu His Glu Tyr Arg Arg Trp Ile Arg Thr Gin His Asn Leu Arg val Val Leu Phe Asp Ile Asp Lys Phe Lys Ser Ile Asn Asp Ser Phe Gly Tyr Thr Ala Gly Asp Lys Ala Leu Ser Ile Ile Ala Arg Thr Ile Lys Lys Glu Leu Arg Asp Ser Asp Thr val Ala Arg Phe Ser Gly Glu Glu Phe Ile Leu Leu Leu Pro Glu Arg Ser Asp Asn Glu Ser Tyr Gin Ile Ile His Gin Ile Gin Leu Asn val Ser Lys Leu Pro Phe Lys Phe Arg Asp Lys Ser Leu Thr Ile Thr Leu ser Ala Ala Ser Ile Arg Phe Met Asp Ser Asp Thr Pro Glu Thr Val Leu Asp Arg Leu Asn Leu Thr Leu Ser Glu Ala Lys His Ile Gly Pro Ser Gin Leu val Trp Lys <210> 31 <211> 339 , <212> PRT
<213> vibrio cholerae <400> 31 Met Met Thr Thr Glu Asp Phe Lys Lys Ser Thr Ala Asn Leu Lys Lys val Val Pro Leu Met Met Lys His His Val Ala Ala Thr Pro Val Asn Tyr Ala Leu Trp Tyr Thr Tyr Val Asp Gln Ala Ile Pro Gln Leu Asn Ala Glu Met Asp Ser Val Leu Lys Asn Phe Gly Leu Cys Pro Pro Ala Ser Gly Glu His Leu Tyr Gln Gln Tyr Ile Ala Thr Lys Ala Glu Thr Asn Ile Asn Gln Leu Arg Ala Asn Val Glu Val Leu Leu Gly Glu Ile Ser Ser Ser Met Ser Asp Thr Leu Ser Asp Thr Ser Ser Phe Ala Asn val Ile Asp Lys Ser Phe Lys Asp Leu Glu Arg Val Glu Gln Asp Asn Leu Ser Ile Glu Glu Val Met Thr Val Ile Arg Arg Leu Val Ser Asp Ser Lys Asp Ile Arg His Ser Thr Asn Phe Leu Asn Asn Gln Leu Asn Ala Ala Thr Leu Glu Ile Ser Arg Leu Lys Glu Gin Leu Ala Lys val Gln Lys Asp Ala Leu Phe Asp Ser Leu Ser Gly Leu Tyr Asn Arg Arg Ala Phe Asp Gly Asp Met Phe Thr Leu Ile His Ala Gly Gln Gln Val Ser Leu Ile Met Leu Asp Ile Asp His Phe Lys Ala Leu Asn Asp Asn Tyr Gly His Leu Phe Gly Asp Gln Ile Ile Arg Ala Ile Ala Lys Arg Leu Gin Ser Leu cys Arg Asp Gly val Thr Ala Tyr Arg Tyr Gly Gly Glu Glu Phe Ala Leu Ile Ala Pro His Lys Ser Leu Arg Ile Ala Arg Gln Phe Ala Glu Ser Val Arg Arg Ser Ile Glu Lys Leu Thr val Lys Asp Arg Arg Ser Gly Gin Ser val Gly Ser Ile Thr Ala Ser Phe Gly val val Glu Lys Ile Glu Gly Asp Ser Leu Glu Ser Leu Ile Gly Arg Ala Asp Gly Leu Leu Tyr Glu Ala Lys Asn Leu Gly Arg Asn Arg val Met Pro Leu <210> 32 <211> 1020 <212> DNA
<213> vibrio cholerae <400> 32 atgatgacaa ctgaagattt caaaaaatcc acggctaact taaaaaaagt cgtaccttta 60 atgatgaaac atcatgtcgc ggccaccccc gtgaactatg ccttgtggta tacctacgtc 120 gaccaagcca ttccgcaact gaatgcggaa atggactctg tattgaaaaa ttttgggctt 180 tgcccacccg cttctggtga acatctttac caacaataca ttgcgaccaa agcagaaacc 240 aatattaatc agttacgtgc gaatgttgag gtacttcttg gtgaaattag cagttcaatg 300 agtgatacgc tcagtgacac cagttccttt gctaatgtga ttgataaaag ctttaaggat 360 ttagagcgcg tcgagcaaga caatctctcg attgaagaag taatgacggt gatccgccgc 420 ttggtgagtg actctaaaga tattcgacac tcaaccaatt tcctaaataa tcaactgaac 480 gcggcaacac tagaaatctc tcgtcttaaa gagcagctgg cgaaagttca gaaagatgct 540 ctgtttgaca gtttatctgg actctataac cgccgagctt ttgatggcga tatgttcacg 600 ctgatccatg caggtcaaca agtcagcctg atcatgctcg acatcgacca cttcaaagcc 660 cttaatgata actatggcca cctgtttggt gaccaaatta tccgtgcgat cgccaaacgt 720 cttcaaagcc tatgccgtga cggcgtgaca gcttatcgtt atggcggtga agagtttgca 780 ctgattgctc cgcacaaatc gctgcgtatt gcacgccagt ttgctgaatc ggtgcgacgt 840 tcaatagaaa agctcaccgt aaaagatcgg cgtagcggtc aatcggtcgg tagcattacc 900 gcttcgtttg gtgtagtaga aaagattgaa ggtgactctt tggagtctct tatcggtcga 960 gcggatggat tgctgtatga agcgaaaaat ctgggccgca atcgagtcat gccgctcttg 1020 <210> 33 <211> 1020 <212> DNA
<213> Vibrio cholerae <400> 33 gtgatgacaa ctgaagattt caaaaaatcc acggctaact taaaaaaagt cgtaccttta 60 atgatgaaac atcatgtcgc ggccaccccc gtgaactatg ccttgtggta tacctacgtc 120 gaccaagcca ttccgcaact gaatgcggaa atggactctg tattgaaaaa ttttgggctt 180 tgcccacccg cttctggtga acatctttac caacaataca ttgcgaccaa agcagaaacc 240 aatattaatc agttacgtgc gaatgttgag gtacttcttg gtgaaattag cagttcaatg 300 agtgatacgc tcagtgacac cagttccttt gctaatgtga ttgataaaag ctttaaggat 360 ttagagcgcg tcgagcaaga caatctctcg attgaagaag taatgacggt gatccgccgc 420 ttggtgagtg actctaaaga tattcgacac tcaaccaatt tcctaaataa tcaactgaac 480 gcggcaacac tagaaatctc tcgtcttaaa gagcagctgg cgaaagttca gaaagatgct 540 ctgtttgaca gtttatctgg actctataac cgccgagctt ttgatggcga tatgttcacg 600 ctgatccatg caggtcaaca agtcagcctg atcatgctcg acatcgacca cttcaaagcc 660 cttaatgata actatggcca cctgtttggt gaccaaatta tccgtgcgat cgccaaacgt 720 cttcaaagcc tatgccgtga cggcgtgaca gcttatcgtt atggcggtga agagtttgca 780 ctgattgctc cgcacaaatc gctgcgtatt gcacgccagt ttgctgaatc ggtgcgacgt 840 tcaatagaaa agctcaccgt aaaagatcgg cgtagcggtc aatcggtcgg tagcattacc 900 gcttcgtttg gtgtagtaga aaagattgaa ggtgactctt tggagtctct tatcggtcga 960 gcggatggat tgctgtatga agcgaaaaat ctgggccgca atcgagtcat gccgctctaa 1020 <210> 34 <211> 461 <212> PRT
<213> vibrio cholerae <400> 34 Met Asp His Arg Phe Ser Thr Lys Leu Phe Leu Leu Leu Met Ile Ala Trp Pro Leu Leu Phe Gly Ser met Ser Glu Ala val Glu Arg Gin Thr Leu Thr Ile Ala Asn Ser Lys Ala Trp Lys Pro Tyr Ser Tyr Leu Asp Glu Gin Gly Gin Pro Ser Gly Ile Leu Ile AS Phe Trp Leu Ala Phe Gly Glu Ala Asn His val Asp Ile Glu Phe Gin Leu met Asp Trp Asn 'Asp Ser Leu Glu Ala val Lys Leu Gly Lys Ser Asp val Gin Ala Gly Leu Ile Arg Ser Ala Ser Arg Leu Ala Tyr Leu Asp Phe Ala Glu Pro Leu Leu Thr Ile Asp Thr Gin Leu Tyr Val His Arg Thr Leu Leu Gly Asp Lys Leu Asp Thr Leu Leu Ser Gly Ala Ile Asn val Ser Leu Gly val val Lys Gly Gly Phe Glu Gin Glu Phe met Gin Arg Glu Tyr Pro Gin Leu Lys Leu Ile Glu Tyr Ala Asn Asn Glu Leu Met Met Ser Ala Ala Lys Arg Arg Glu Leu Asp Gly Phe val Ala AS Thr Gin val Ala Asn Phe Tyr Ile val val Ser Asn Gly Ala Lys Asp Phe Thr Pro val Lys Phe Leu Tyr Ser Glu Glu Leu Arg Pro Ala Val Ala Lys Gly Asn Arg Asp Leu Leu Glu Gin Val Glu Gin Gly Phe Ala Gin Leu Ser Ser Asn Glu Lys Asn Arg Ile Leu Ser Arg Trp val His Ile Glu Thr Ile Tyr Pro Arg Tyr Leu Met Pro Ile Leu Ala Ser Gly Leu Leu Leu Ser Ile val Ile Tyr Thr Leu Gin Leu Arg Arg Thr Val Arg Leu Arg Thr Gin Gin Leu Glu Gill Ala Asn Gin Lys Leu Ser Tyr Leu Ala Lys Thr Asp ser Leu Thr Asp Ile Ala Asn Arg Arg Ser Phe Phe Glu His Leu Glu Ala Glu Gin Thr Arg Ser Gly Ser Leu Thr Leu Met val Phe Asp Ile Asp AS Phe Lys Thr Ile Asn Asp Arg Phe Gly His Gly Ala Gly Asp Asn Ala Ile Cys Phe val val Gly Cys val Arg Gin Ala Leu Ala Ser Asp Thr Tyr Phe Ala Arg Ile Gly Gly Glu Glu Phe Ala Ile val Ala Arg Gly Lys Asn Ala Glu Glu Ser Gin Gin Leu Ala Glu Arg Ile Cys Gin Arg Val Ala Glu Lys Lys Trp Val Val Asn Ala Gin His Ser Leu Ser Leu Thr Ile Ser Leu Gly Cys Ala Phe Tyr Leu HiS Pro Ala Arg Pro Phe Ser Leu His Asp Ala Asp Ser Leu Met Tyr Glu Gly Lys Arg Asn Gly Lys Asn Gin val Val Phe Arg Thr Trp Ser <210> 35 <211> 1386 <212> DNA
<213> vibrio cholerae <400> 35 atggatcatc gcttttcgac caaactgttt ctgcttctca tgattgcttg gccgctttta 60 ttcggatcaa tgagtgaggc tgtagagcgc caaaccttga ctattgccaa ctcaaaagca 120 tggaaaccct attcttattt ggatgaacag ggacagcctt ctggcatatt gattgatttt 180 tggttggctt ttggtgaagc gaatcatgtc gatattgaat tccaactgat ggattggaat 240 gattccctag aagcggtgaa gcttggcaaa tccgatgttc aagctggttt gatccgttct 300 gcttcaagat tagcgtatct cgattttgca gaacctttac tgacaatcga tacacaactc 360 tacgtacacc gcacgttatt gggcgataaa ttggatacgc tgctatcggg ggCcattaac 420 gtctcattag gtgtagtaaa agggggattt gaacaagagt tcatgcaacg agaatatcct 480 caacttaagt tgattgagta cgccaacaat gaattgatga tgtctgcagc aaagcgacga 540 gaattagatg gttttgtggc cgatactcag gtcgccaatt tctatatagt ggtttccaat 600 ggcgcgaaag attttacgcc agtgaagttt ctttattcag aggaattacg tccagcggtc 660 gccaaaggca atagggattt attagagcaa gtagagcagg ggtttgcaca attaagtagc 720 aatgagaaaa accgtatttt aagtcgatgg gttcatattg aaacgattta tccacgttac 780 ttaatgccga ttctcgcttc aggtctctta ctcagtatcg ttatttatac tcttcagcta 840 cggcgtaccg ttcgattgcg aacacagcaa cttgaagaag ccaatcaaaa actctcctat 900 ttagcgaaaa cggatagctt gacggacatt gctaatcgcc gttcgttttt tgaacatctt 960 gaagcggaac aaacacgatc aggcagctta acgttgatgg tttttgatat tgatgacttc 1020 aaaaccatta acgatcgctt tgggcatggc gcaggagata atgccatctg tttcgtggtt 1080 gggtgtgtgc gacaagcttt agcatcggat acctactttg caaggattgg tggtgaagag 1140 tttgctattg tagcgcgtgg taaaaatgca gaagagtcgc agcagttagc tgagcgaatt 1200 tgccaacgag ttgcagaaaa aaagtgggta gtgaatgccc aacactctct gtcactcacc 1260 atcagcctag gctgtgcatt ttacctacac ccagctcggc cattcagttt gcacgatgcc 1320 gatagcttaa tgtacgaagg aaagcggaat ggaaagaacc aggttgtctt tcgtacctgg 1380 tcataa 1386 <210> 36 <211> 420 <212> PRT
<213> Vibrio cholerae <400> 36 Met AS His Arg Phe Ser Thr Lys Leu Phe Leu Leu Leu Met Ile Ala Trp Pro Leu Leu Phe Gly Ser Met Ser Glu Ala Val Glu Arg Gln Thr Leu Thr Ile Ala Asn Ser Lys Ala Trp Lys Pro Tyr Ser Tyr Leu Asp Glu Gln Gly Gln Pro Ser Gly Ile Leu Ile Asp Phe Trp Leu Ala Phe Gly Glu Ala Asn His Val Asp Ile Glu Phe Gln Leu Met Asp Trp Asn Asp Ser Leu Glu Ala Val Lys Leu Gly Lys Ser Asp Val Gln Ala Gly Leu Ile Arg Ser Ala Ser Arg Leu Ala Tyr Leu Asp Phe Ala Glu Pro Leu Leu Thr Ile Asp Thr Gln Leu Tyr val His Arg Thr Leu Leu Gly Asp Lys Leu Asp Thr Leu Leu Ser Gly Ala Ile Asn Val Ser Leu Gly Val val Lys Gly Gly Phe Glu Gln Glu Phe Met Gln Arg Glu Tyr Pro Gln Leu Lys Leu Ile Glu Tyr Ala Asn Asn Glu Leu Met Met Ser Ala Ala Lys Arg Arg Glu Leu Asp Gly Phe Val Ala Asp Thr Gln Val Ala Asn Phe Tyr Ile Val Val Ser Asn Gly Ala Lys Asp Phe Thr Pro Val Lys Phe Leu Tyr Ser Glu Glu Leu Arg Pro Ala val Ala Lys Gly Asn Arg Asp Leu Leu Glu Gin val Glu Gin Gly Phe Ala Gin Leu Ser Ser Asn Glu Lys Asn Arg Ile Leu Ser Arg Trp Val His Ile Glu Thr Ile Tyr Pro Arg Tyr Leu Met Pro Ile Leu Ala Ser Gly Leu Leu Leu Ser Ile Val Ile Tyr Thr Leu Gin Leu Arg Arg Thr Val Arg Leu Arg Thr Gin Gin Leu Glu Glu Ala Asn Gin Lys Leu Ser Tyr Leu Ala Lys Thr Asp Ser Leu Thr Asp Ile Ala Asn Arg Arg Ser Phe Phe Glu His Leu Glu Ala Glu Gin Thr Arg Ser Gly Ser Leu Thr Leu Met Val Phe Asp Ile Asp Asp Phe Lys Thr Ile Asn Asp Arg Phe Gly His Gly Ala Gly Asp Asn Ala Ile Cys Phe Val Val Gly Cys Val Arg Gin Ala Leu Ala Ser Asp Thr Tyr Phe Ala Arg Ile Gly Gly Glu Glu Phe Ala Ile Val Ala Arg Gly Lys Asn Ala Glu Glu Ser Gin Gin Leu Ala Glu Arg Ile Cys Gin Arg Val Ala Glu Lys Lys Trp Val Val Asn Ala Gin His Ser Leu Ser Leu Thr <210> 37 <211> 1239 <212> DNA
<213> Vibrio cholerae <400> 37 ttagctagcg actttgacac aattgcgccc agcttgcttc gctttataaa gtgccccatc 60 cgctgctttg agtgcctcaa taggatggcg gtacagctca gaatcacaca cgccaatgct 120 gatggtaata gtgacaatgt cactgttact ttttcggctg cgtttttttg caccttcagc 180 atgacttttc gggcgctggt tggtgtcacg aatcaccaac tcgtaggact caatatcctg 240 ccgtaaggcc tcgatgaaag gcaaaacctc ctttgccaat tttcctttgt aaataatcga 300 gaactcctca ccaccatagc ggtaaactcg tgctttaccg ttgatttcac gtaatcgaga 360 ggcaaccagt cttaatacat cgtcccccgt atcatgcccg taagtatcgt taaacttctt 420 gaaatggtcg acatcgagca tagcgagggt aaattttcga cctatatgtt ttaaatcctg 480 atcaagcgct tgccgaccag gaatttgggt gagtgggtcg ttaaatgcca tctcatagcc 540 cgcggaaatg aggtaaacca gaataagcag cccagataag gtaaacatga tggtggaaat 600 ataaggcaca tgaaacagca caaacgcatt catgctcaat acaatcgaac tataaaccac 660 aacatcaaga atttgattgc gcgttaatac cgagatagca gcaatacctg cgagtgcgac 720 aagataggca acaaccacca agggtaagcg agaaatttgc ggtacaacga aaaatattcc 780 ctcggtgagg ctggaatggt ctgtttcacc tatgtgtagc tgggtcagcc aagcccaaaa 840 gatgaacagc aataaaatag ccaagtaact gagaaaggat ttgctgaata atccagcatt 900 cttgtaggcg taaggtaaaa aacaggccac aggcaaaagc aagctcagca taatgagttc 960 aagcatggtg gaattgacgg ttaaaggcgt ttgaagtcga atttggatca accagtaagc 1020 cagtaacatc gtcatcgcta ccatggcgat tctgctttgt ttaaaaatgt gagcaacggt 1080 tagcgcaatc aaaaagagaa tgtaggggag gttgaccgcc atgcctaagt tagactttat 1140 caccaatacc acattgctca agcctagcca aatggctacc agcagcaata gaggaaaacc 1200 gaaacggaac caaggtgaag taacaaagct agaagacat 1239 <210> 38 <211> 453 <212> PRT
<213> vibrio cholerae <400> 38 Met Ser Ser Ser Phe val Thr Ser Pro Trp Phe Arg Phe Gly Phe Pro Leu Leu Leu Leu val Ala Ile Trp Leu Gly Leu Ser Asn val val Leu val Ile Lys Ser Asn Leu Gly met Ala val Asn Leu Pro Tyr Ile Leu Phe Leu Ile Ala Leu Thr val Ala His Ile Phe Lys Gln Ser Arg Ile Ala met val Ala Met Thr met Leu Leu Ala Tyr Trp Leu Ile Gin Ile Arg Leu Gin Thr Pro Leu Thr val Asn Ser Thr Met Leu Glu Leu Ile Met Leu Ser Leu Leu Leu Pro val Ala Cys Phe Leu Pro Tyr Ala Tyr Lys Asn Ala Gly Leu Phe Ser Lys Ser Phe Leu Ser Tyr Leu Ala Ile Leu Leu Leu Phe Ile Phe Trp Ala Trp Leu Thr Gin Leu His Ile Gly Glu Thr Asp His Ser Ser Leu Thr Glu Gly Ile Phe Phe val val Pro Gin Ile Ser Arg Leu Pro Leu val val val Ala Tyr Leu val Ala Leu Ala Gly Ile Ala Ala Ile Ser Val Leu Thr Arg Asn Gin Ile Leu Asp Val val Val Tyr Ser Ser Ile Val Leu Ser Met Asn Ala Phe Val Leu Phe His Val Pro Tyr Ile Ser Thr Ile met he Thr Leu Ser Gly Leu Leu Ile Leu val Tyr Leu Ile Ser Ala Gly Tyr Glu Met Ala Phe Asn Asp Pro Leu Thr Gin Ile Pro Gly Arg Gin Ala Leu Asp Gin Asp Leu Lys His Ile Gly Arg Lys Phe Thr Leu Ala met Leu Asp Val Asp His Phe Lys Lys Phe Asn Asp Thr Tyr Gly His Asp Thr Gly Asp Asp val Leu Arg Leu Val Ala Ser Arg Leu Arg Glu Ile Asn Gly Lys Ala Arg Val Tyr Arg Tyr Gly Gly Glu Glu Phe Ser Ile Ile Tyr Lys Gly Lys Leu Ala Lys Glu Val Leu Pro Phe Ile Glu Ala Leu Arg Gin Asp Ile Glu Ser Tyr Glu Leu val Ile Arg Asp Thr Asn Gin Arg Pro Lys Ser His Ala Glu Gly Ala Lys Lys Arg Ser Arg Lys Ser Asn Ser Asp Ile Val Thr Ile Thr Ile Ser Ile Gly val Cys Asp Ser Glu Leu Tyr Arg His Pro Ile Glu Ala Leu Lys Ala Ala Asp Gly Ala Leu Tyr Lys Ala Lys Gln Ala Gly Arg Asn Cys Val Lys Val Ala Ser Ile Ser Leu Gly Cys Ala Phe Tyr Leu His Pro Ala Arg Pro Phe Ser Leu His Asp Ala Asp Ser Leu Met Tyr Glu Gly Lys Arg Asn Gly Lys Asn Gin Val Val Phe Arg Thr Trp Ser <210> 39 <211> 1884 <212> DNA
<213> Vibrio cholerae <400> 39 atgtacacct cagcccgtaa atatttcata caatttgcca ttgttgcgtt tgtacttggt 60 ttcattccta cactgtattt catacatgct gctagccagc ttgagactca agcggtcagc 120 agcgttgaaa aacagactcg cttacagctt gagttcagtc agcatgactt gttacgaatg 180 ctggaaagca cacaccaagc cacccagctg ttagctaaaa atgacctttt attcacggct 240 gtcaccacac caagcaaaga agcactcagt caactcaaaa cattgtggga tgtgacgtta 300 agatcgcaag cgattttctc ttcattcaga ttgctggata gacaaggaaa agaacaactt 360 aaagcgattt acgatgggca ccaagtcacc tttgttgaat ctgctcaaac gacagatccg 420 ttcagccagc aaattgtggc tcaatacgcc caactcacga cgcctcaagt ttgggcaacg 480 caagtcgcga tgtcagcaga tacgccttct ggtatgctgc cgacctttcg ttttgtgacg 540 ggtattgagc atcaaggcca acggcaaggt tttcttgtcg tgacggtgaa gctacagtct 600 ctctatcaac gtctctcttt tatttatgat cagtttgatt caccggatat tttgaattcg 660 gcaggagaat tactgctcag tgaacacaag ccatccggta cacgttcaac ctcttcactc 720 cacttttcag cccaacaccc agagctttgg caaaaaatcc aactcaacca acaaggcttt 780 gctctatcca atcaaacctg gtttagctat atcaaagtgg atctcagttc tgtcttacct 840 gactttaaac ctttggtatt ggtactgcgc atcaataagg cagaaataga taagacctac 900 gcaaatgcgc gctgggcact gatgagtcaa gcggtgacag tgttatcgct actctctatc 960 attgcggctg gatttgcggc atggaacatc aaccatttaa aaaatagcct tgacagtaaa 1020 ttggcicgag cagcgatgga tggcatgtca gcggtggtca ttaccgaccg ccagaatcgc 1080 atcatcaaag taaacaacga atttacccgc ctaagtggtt acacttttga agatgtcaaa 1140 ggtaagcagc cgtccatttt tgcttctgga ttacacaaag tcgaattcta tatgcagatg 1200 tggaaagctc tgcaagacaa tggcgtatgg gaaggtgaag tgatcaacaa acgcaaagat 1260 ggcgaaagca tcaccgaaat tctccgtatt caaagcatcc gcgatgaaga caatgtcatt 1320 caattctacg ttgcctcttt tgtggatatt tcacatcgca aggcgctgga gaatcgcctg 1380 cgtgagctga gcgaaaaaga tgcgttaacc gatttgtgga atcgacgtaa attcgatcaa 1440 accatctctt tagagtgcgc taagcgtcgc cgttatcccg atcaagccca gagctgcctt 1500 gctatcattg atatcgacca ctttaaacgc attaacgaca aattcggaca caacgaaggg 1560 gacctagtgt tacggaccgt tgcgaaaggc atccaagatc agttacggga atcggatttt 1620 atcgcacgga ttggcggaga agagtttgcc attattttcc cctacacttc cattgaagaa 1680 gccgaacaag tacttaaccg cgtacgcctg catatcgctt cattacacca tcaacaagtg 1740 accctaagtg gtggtgttac cgatgtttgc acatcacccg accaaagcta caaaagagcc 1800 gatctggctt tatatgaatc caaaacatcg ggacgcaacc aaatatcagt actcaccgcc 1860 atggaaatgc atcactttgc gtga 1884 <210> 40 <211> 627 <212> PRT
<213> Vibrio cholerae <400> 40 met Tyr Thr Ser Ala Arg Lys Tyr Phe Ile Gin Phe Ala Ile Val Ala Phe Val Leu Gly Phe Ile Pro Thr Leu Tyr Phe Ile His Ala Ala Ser Gin Leu Glu Thr Gin Ala Val Ser Ser Val Glu Lys Gin Thr Arg Leu Gin Leu Glu Phe Ser Gin His Asp Leu Leu Arg Met Leu Glu Ser Thr His Gin Ala Thr Gin Leu Leu Ala Lys Asn Asp Leu Leu Phe Thr Ala Val Thr Thr Pro Ser Lys Glu Ala Leu Ser Gin Leu Lys Thr Leu Trp Asp val Thr Leu Arg Ser Gin Ala Ile Phe Ser Ser Phe Arg Leu Leu Asp Arg Gin Gly Lys Glu Gin Leu Lys Ala Ile Tyr Asp Gly His Gin . , Val Thr Phe val Glu Ser Ala Gin Thr Thr Asp Pro Phe Ser Gin Gin Ile val Ala Gin Tyr Ala Gin Leu Thr Thr Pro Gin val Trp Ala Thr Gin Val Ala met Ser Ala Asp Thr Pro Ser Gly Met Leu Pro Thr Phe Arg Phe Val Thr Gly Ile Glu His Gin Gly Gin Arg Gin Gly Phe Leu val val Thr Val Lys Leu Gin Ser Leu Tyr Gin Arg Leu Ser Phe Ile Tyr Asp Gin Phe Asp Ser Pro Asp Ile Leu Asn Ser Ala Gly Glu Leu Leu Leu Ser Glu His Lys Pro Ser Gly Thr Arg Ser Thr Ser Ser Leu His Phe Ser Ala Gin His Pro Glu Leu Trp Gin Lys Ile Gin Leu Asn Gin Gin Gly Phe Ala Leu Ser Asn Gin Thr Trp Phe Ser Tyr Ile Lys Val Asp Leu Ser Ser Val Leu Pro AS Phe Lys Pro Leu Val Leu Val Leu Arg Ile Asn Lys Ala Glu Ile Asp Lys Thr Tyr Ala Asn Ala Arg Trp Ala Leu Met Ser Gin Ala Val Thr Val Leu Ser Leu Leu Ser Ile Ile Ala Ala Gly Phe Ala Ala Trp Asn Ile Asn His Leu Lys Asn Ser Leu Asp Ser Lys Leu Ala Arg Ala Ala Met Asp Gly Met Ser Ala Val val Ile Thr Asp Arg Gin Asn Arg Ile Ile Lys Val Asn Asn Glu Phe Thr Arg Leu Ser Gly Tyr Thr Phe Glu Asp Val Lys Gly Lys Gln Pro Ser Ile Phe Ala Ser Gly Leu His Lys Val Glu Phe Tyr met Gin Met Trp Lys Ala Leu Gin Asp Asn Gly Val Trp Glu Gly Glu Val Ile Asn Lys Arg Lys Asp Gly Glu Ser Ile Thr Glu Ile Leu Arg Ile Gin Ser Ile Arg Asp Glu Asp Asn Val Ile Gin Phe Tyr val Ala Ser Phe Val Asp Ile Ser His Arg Lys Ala Leu Glu Asn Arg Leu Arg Glu Leu Ser Glu Lys Asp Ala Leu Thr Asp Leu Trp Asn Arg Arg Lys Phe Asp Gin Thr Ile Ser Leu Glu Cys Ala Lys Arg Arg Arg Tyr Pro Asp Gin Ala Gin Ser Cys Leu Ala Ile Ile Asp Ile Asp His Phe Lys Arg Ile Asn Asp Lys Phe Gly His Asn Glu Gly Asp Leu Val Leu Arg Thr Val Ala Lys Gly Ile Gin Asp Gin Leu Arg Glu Ser Asp Phe Ile Ala Arg Ile Gly Gly Glu Glu Phe Ala Ile Ile Phe Pro Tyr Thr Ser Ile Glu Glu Ala Glu Gin val Leu Asn Arg Val Arg Leu His Ile Ala Ser Leu His His Gin Gin Val Thr Leu Ser Gly Gly Val Thr Asp Val Cys Thr Ser Pro Asp Gin Ser Tyr Lys Arg Ala Asp Leu Ala Leu Tyr Glu Ser Lys Thr Ser Gly Arg Asn Gin Ile Ser Val Leu Thr Ala Met Glu Met His His Phe Ala <210> 41 <211> 1884 <212> DNA
<213> Vibrio cholerae <400> 41 atggcaccga tcctttcaca ctcgatcccg atcccttcta gcatgcaggc aaattggcag 60 apJaLog) opm.A <ETZ>
Did <ZTZ>
LZ9 <TTZ>
Zt <OTZ>
EE16 36613136)e 336eEE631) 0981 6ee)1))3E1 11616EE616 ED)361P/DD EEE)661.E1E 1.1111.)66ee )36633.61.E6 008T 3611666E)E E661)11E31 16E)6E6EE) 6E363E6)16 E611E166Ey 661.6616616 OVLT DePP11)PP6 1DPPPPRP6) 6P161P)61) 61P1D116D6 16)1PPP6PP Dap6Appp6 0891 D6PDPDEP6D 1P6ED6P661 P11116EPPP 1P611P6PED 1U)661P611 1E)))1.1EEE
OZ91 EEEE1)1.E16 E)111.6)113 6)E11.666)E 166a111e61 U6D1DED1P1 1E3E616)66 09S1 Pl1PDPDPDP 1DP6DDETD1 Da1P1P616P 6)6)3E63)6 p61.E6)1631 E61)36616e 00ST PP6D)PDITP P661DPP661 16pplED6D6 133D11361.1 pa61D6D661 )6131p6p6D
OttT lap5D6E6D 1611.1.36616 PDPPEPPD1) 6PDP163)11 D1D161EPD1 E636)1E161.
HET 111DDPPDDD 616166)6P1 16611P616P 3)6DAPPPP 111D6PPDDE D611116111 OZET 1.E)666616E E6114D1P11 )1661EERED 6E6E)661.E1 E)11E61133 epu6luE))1.
09ZT 111EE61.666 1161663)1E 36EE661.16) 611611E366 '61.36166)1 6661)636E) 00Z1 3)EEEE61E) 6ED16EPP1) DPP))11DP1 D/D61)61)1 P6DUPDPUD6 )616636Er) OKI 613E111E1) epee6611EE pleeee)66) e361.eopeel e63 6u 6)))1)13e) 0801 1.1.1411616E EEDEI.E6663 1)E66EE161 6)661161E) 6E36E31633 61E3)6361) OZOT ulp6E36E36 )31E636)E3 EEE616))6e 1133)66161 6611P1P11) DDPED1DDVD
096 1163)36136 666E663E61 61.11Ep36)6 EE)6661)6) E311.EE111.6 )1E3)6111) 006 6EDITEElle PD)DPDP6D1 )67)1P)))6 UPPP11D1P6 1)6DP6111E PDDP6D1DPP
0178 1.3)3631)6) pr61.3E1.1EE 66666E663e 61663)1.661. 13E6)6E626 3p1.1.6pAue 08L E136161E61 p666111)3E 33E116661) 66D6D66PDP 11EPDADDD 61PED)6111 OZL 166)1P1ED1 1P6161D6D1 D6P6PP6DPD PP66D6611P PD1D661)6P D61e6D6)1) 099 6E36E36E66 11E3663E36 plEE613111 )6613E6661 4)3E63E316 E)636E31.E1 009 6P611P6PD6 PDPP6PD63) 616))6DPUP 1P6D1P6P6P ED1P3D1D1D 1PPDDPP61) OtS 6D1))6D1P) 6PPP)61UPP P1P)16)116 PRED11DED6 111P6PPDDP P6aePPD611 0817 6116)EEE63 6)EenE11.1. 3DDPPPP11) 6E D66. 1P6D1PP61) 6111)DPPEE
OZt P1P611EPD6 eD1P6DDP16 P11)1116P) D6D1D6PED1 6D1P6DDUD1 61616111)1 09E E661.1111)6 E61663EEE) 3661EE)611 EnE163661 613E11)636 )61E366611 00E EE661)1E6E DDDPP1P66E P661611ED3 DP6D1D)P6D D6DPP16PD1 6P11611P1P
OtZ 6PDAD1D D166161) PPP61611P1 611)66PDPD 66P11P6DPE 6DDP61E)66 081 )16PPDDE1P DD1PPDP616 ED66)1616P DDP1611116 161P611)1E 6)PD1PDAD
OZT E11.1.6)61E3 1E61)33E33 6e31)16EET 613616pE6) )661)61.3)E e)1.361E6E3 , <400> 42 Met Ala Pro Ile Leu Ser His Ser Ile Pro Ile Pro Ser Ser met Gin Ala Asn Trp Gin Gin Met Leu Asn Leu Leu Ala Glu Val Leu Lys Val Ser Ala Thr Leu Ile Met Arg Leu Arg His His Asp Leu Asp Val Phe Cys Thr Ser Val Gly Ser Asp Asn Pro Tyr Gin Val Gly Met Thr Glu Arg Leu Gly Thr Gly Leu Tyr Cys Glu Thr val Val Asn Thr Arg Gin Ile Leu Leu Val Ser Asn Ala Asp Leu Asp Pro Leu Trp Lys Asp Asn Pro Asp Leu Glu Leu Gly Met Arg Ala Tyr Cys Gly Val Pro Leu Gin Trp Pro Asn Gly Glu Leu Phe Gly Ser Leu Cys Val Thr Asp Arg Gin Ala Arg Gin Phe Leu Ser Thr Asp Gin Gin Leu Ile Lys Thr Phe Ala Glu Ser Ile Glu Ala Gin Leu Lys Thr Leu Tyr Gin Arg Glu Thr Leu Leu Gin Met Asn Gin Asp Leu His Phe Lys val Arg His Lys Met Gin Ser Ile Ala Ser Leu Asn Gin Ser Leu His Gin Glu Ile Asp Lys Arg Arg Ala Ala Glu Gin Gin Ile Glu Tyr Gin Arg Ser His Asp Leu Gly Thr Gly Phe Leu Asn Arg Thr Ala Leu Glu Gin Gin Leu Ala Met Gin Leu Ala Gln Leu Ala Glu His Glu Glu Leu Ala Val Ile His Ile Gly Phe Ala Asn Ala Arg Gin Leu Gin Ala Arg Leu Gly Tyr HiS Leu Trp Asp Asp val Leu Lys Gin Leu Arg Glu Arg Leu Gly Pro val Thr Glu , , Gly Glu Leu Leu Thr Ala Arg Pro Asn Ser Thr Asn Leu Thr Leu Ile Leu Lys Ala His Pro Leu Asp Thr Gin Leu Asn Gin Leu Cys His Arg Leu Ile His Ala Gly Gin Ala Gin Phe Val Thr Glu Gly Leu Pro Val His Leu Asn Pro Tyr Ile Gly Val Ala Leu Ser Arg Glu Thr Arg Asp Pro Gin Gin Leu Leu Arg His Ala Val Ser Ser Met Leu Ala Cys Lys Asp Ser Gly Tyr Lys Val Phe Phe His Ser Pro Ala Leu Ala Asp Asn His Ala Arg Gin Asn Gin Leu Glu Asn Tyr Leu Leu Gin Ala Val Arg Asn Asn Asp Leu Leu Leu Tyr Phe Gin Pro Lys Val Ser Met Lys Thr Gin Arg Trp Val Gly Ala Glu Ala Leu Leu Arg Trp Lys His Pro Val Leu Gly Glu Phe Ser Asn Glu Thr Leu Ile His Met Ala Glu Gin Asn Gly Leu Ile Phe Glu Val Gly His Phe Val Leu His Gin Ala Leu Lys Ala Ala Ser Asp Trp Leu Ala Val Cys Pro Thr Phe Cys Ile Ala Ile Asn Val Ser Ser Val Gin Leu Lys Asn Ser Gly Phe Val Glu Gin Ile Arg Asp Leu Leu Ala Leu Tyr Cys Phe Pro Ala His Gin Leu Glu Leu Glu Ile Thr Glu Ser Gly Leu Ile Val Asp Glu Pro Thr Ala Ser Asp Ile Leu Asn Arg Leu His Thr Leu Gly Val Thr Leu Ser Leu Asp Asp Phe Gly Thr Gly Tyr Ala Ser Phe Gin Tyr Leu Lys Lys Phe Pro Phe Asp Gly Ile Lys Ile Asp Lys Ser Phe Met Glu Gin Ile Glu His Ser Glu Ser Asp Gin Glu Ile Val Arg Ser met Leu His Val Ala Lys Lys Leu Asn Leu Asn Val Val Val Glu Gly Ile Glu Ser Thr Gin Gin Glu Gin Phe Ile Leu Glu Gin Gly Cys Asp Val Gly Gin Gly Phe Leu Tyr Gly Lys Pro Met Pro Ser Glu Val Phe Thr Leu Lys Leu Glu Ser His Ala Leu Ala <210> 43 <211> 981 <212> DNA
<213> Vibrio cholerae <400> 43 ttagaaaagt tcaacgtcat cagaaaatgg ccgttgcgcg ctggcaattt taccgttctc 60 acacagctgt tcatagcagt gcacctgatt ccgaccatgc tctttggcgt aatacaacgc 120 tttatcggca tggtcgagaa tggtaggtaa atagtcaccc ggcctgagtg agcaaaaacc 180 agcgctgaag ctcagttcac cgattctcgg gaagttatgg cgtcggatct gttgacggaa 240 gccatccaac tgttgcttga tttgtggctc attaccgctt gaaaaaataa tcacgaactc 300 ttcaccacca aagcgaaata gttgagaaga cggtccgaaa tagtgctgca tctgctgagc 360 gaacataagc agaatttcat caccaatcat gtgtccgaag tgatcattga tcgctttaaa 420 atggtcaata tccaacatcg cgatccagag tttgtgattc tcttctgtcg agggattgat 480 ggcaaaggtg tggcgcaatc ggtcttctaa cgttcgacga ttgagtaatc cggtcagctt 540 atcgcgttca ctctcatgca aaatcaccgt gtaattacgg taaattttcg caaatccgtt 600 gatcaacatg cgataaggtt caggatcttt attgaggatt aagcacagct ctgcggaaaa 660 gtgttcttct atcggaatcg ggcaaaagca ttgatattgg ccattcgctt gttgggaaaa 720 cgccatttcc gattgagagt gctggtaacc attgtcggca catacttggt cgtattgcca 780 ctggtactcc tttttacctg cagcattttt ggtaataatt aaacgtgcca ccataagggt 840 tgaacgtcca agatggtgaa ataaggtcgc cgtggagagc ggtaacaatt cagacaaggt 900 cgccaaaata ctgtaactga gtgccagcga atttttctgc tcagtaattt caataaccga 960 ctcaagcact ttgtcattca t 981 <210> 44 ' <211> 326 <212> PRT
<213> Vibrio cholerae <400> 44 Met Asn Asp Lys Val Leu Glu Ser Val Ile Glu Ile Thr Glu Gin Lys Asn Ser Leu Ala Leu Ser Tyr Ser Ile Leu Ala Thr Leu Ser Glu Leu Leu Pro Leu Ser Thr Ala Thr Leu Phe His His Leu Gly Arg Ser Thr Leu met Val Ala Arg Leu Ile Ile Thr Lys Asn Ala Ala Gly Lys Lys Glu Tyr Gin Trp Gin Tyr Asp Gin Val Cys Ala Asp Asn Gly Tyr Gin His Ser Gin Ser Glu Met Ala Phe Ser Gin Gin Ala Asn Gly Gin Tyr Gin Cys Phe Cys Pro Ile Pro Ile Glu Glu His Phe Ser Ala Glu Leu Cys Leu Ile Leu Asn Lys Asp Pro Glu Pro Tyr Arg Met Leu Ile Asn Gly Phe Ala Lys Ile Tyr Arg Asn Tyr Thr Val Ile Leu His Glu Ser Glu Arg Asp Lys Leu Thr Gly Leu Leu Asn Arg Arg Thr Leu Glu Asp Arg Leu Arg His Thr Phe Ala Ile Asn Pro Ser Thr Glu Glu Asn His Lys Leu Trp Ile Ala Met Leu Asp Ile Asp His Phe Lys Ala Ile Asn Asp His Phe Gly His Met Ile Gly Asp Glu Ile Leu Leu met Phe Ala Gin Gin Met Gin His Tyr Phe Gly Pro Ser Ser Gin Leu Phe Arg Phe Gly Gly Glu Glu Phe Val Ile Ile Phe Ser Ser Gly Asn Glu Pro Gin Ile Lys Gin Gin Leu Asp Gly Phe Arg Gin Gin Ile Arg Arg His Asn Phe Pro Arg Ile Gly Glu Leu Ser Phe Ser Ala Gly Phe Cys Ser Leu Arg Pro Gly Asp Tyr Leu Pro Thr Ile Leu Asp His Ala AS Lys Ala Leu Tyr Tyr Ala Lys Glu His Gly Arg Asn Gin Val His Cys Tyr Glu Gin Leu Cys Glu Asn Gly Lys Ile Ala Ser Ala Gin Arg Pro Phe Ser Asp Asp Val Glu Leu Phe <210> 45 <211> 927 <212> DNA
<213> Vibrio cholerae <400> 45 atgatagaac ttaatagaat tgaagagctt tttgataacc aacagttctc cttgcacgaa 60 ctcgtgttga acgaactggg agtctatgtc ttcgtcaaaa atcgccgcgg cgagtatctc 120 tatgctaacc ctctgactct aaagttgttt gaagcggatg cacaatcgtt gtttggcaag 180 accgatcacg atttttttca tgatgatcaa ctcagtgata tcttggcggc cgatcaacag 240 gtgtttgaaa ctcgtctctc ggttatccat gaagaacgag ccatcgccaa atccaatggt 300 ttggttcgga tttatcgcgc agtcaaacac cctatcttgc accgagtgac aggcgaagtg 360 attgggctga ttggagtttc aaccgatatc accgatatcg tggaactgcg tgagcagcta 420 tatcagctcg ccaataccga ttctttaact cagctgtgta atcggcgtaa attgtgggcc 480 gattttcgcg ccgccttcgc tcgcgcaaaa cgtttaagac agccgttaag ttgcatctct 540 atcgatattg ataatttcaa actgatcaat gaccaatttg gtcacgataa aggtgatgaa 600 gtcctgtgtt ttctcgccaa actatttcag agcgtcatct ctgaccatca tttttgtggt 660 cgtgtgggag gtgaagagtt catcatcgtt ttggaaaata cgcacgtaga gacggctttt 720 catttggctg aacagatccg ccaacgtttt gcagagcatc cgttctttga acaaaacgag 780 cacatctacc tctgtgcggg ggtttccagc ttgcatcatg gtgatcatga cattgccgat 840 atttatcgac gctccgatca agcactgtat aaagccaagc gtaatggtcg taaccgttgc 900 tgtatctatc gccaatccac agaataa 927 <210> 46 <211> 308 <212> PRT
<213> Vibrio cholerae <400> 46 Met Ile Glu Leu Asn Arg Ile Glu Glu Leu Phe Asp ASn Gin Gin Phe , Ser Leu His Glu Leu val Leu Asn Glu Leu Gly Val Tyr val Phe Val Lys Asn Arg Arg Gly Glu Tyr Leu Tyr Ala Asn Pro Leu Thr Leu Lys Leu Phe Glu Ala Asp Ala Gin Ser Leu Phe Gly Lys Thr Asp His Asp Phe Phe His AS Asp Gin Leu Ser Asp Ile Leu Ala Ala Asp Gin Gin val Phe Glu Thr Arg Leu Ser val Ile His Glu Glu Arg Ala Ile Ala Lys Ser Asn Gly Leu Val Arg Ile Tyr Arg Ala Val Lys His Pro Ile Leu His Arg Val Thr Gly Glu Val Ile Gly Leu Ile Gly Val Ser Thr Asp Ile Thr Asp Ile Val Glu Leu Arg Glu Gin Leu Tyr Gin Leu Ala Asn Thr Asp Ser Leu Thr Gin Leu Cys Asn Arg Arg Lys Leu Trp Ala Asp Phe Arg Ala Ala Phe Ala Arg Ala Lys Arg Leu Arg Gin Pro Leu Ser Cys Ile Ser Ile AS Ile Asp Asn Phe Lys Leu Ile Asn Asp Gin Phe Gly His Asp Lys Gly Asp Glu val Leu Cys Phe Leu Ala Lys Leu Phe Gin Ser Val Ile Ser Asp His His Phe cys Gly Arg val Gly Gly Glu Glu Phe Ile Ile Val Leu Glu Asn Thr His Val Glu Thr Ala Phe His Leu Ala Glu Gin Ile Arg Gin Arg Phe Ala Glu His Pro Phe Phe Glu Girl Asn Glu His Ile Tyr Leu Cys Ala Gly Val Ser Ser Leu His His Gly Asp His Asp Ile Ala Asp Ile Tyr Arg Arg Ser Asp Gin Ala Leu Tyr Lys Ala Lys Arg Asn Gly Arg Asn Arg Cys Cys Ile Tyr Arg Gin Ser Thr Glu <210> 47 <211> 1017 <212> DNA
<213> Vibrio cholerae <400> 47 atgacaactg aagatttcaa aaaatccacg gctaacttaa aaaaagtcgt acctttaatg 60 atgaaacatc atgtcgcggc cacccccgtg aactatgcct tgtggtatac ctacgtcgac 120 caagccattc cgcaactgaa tgcggaaatg gactctgtat tgaaaaattt tgggctttgc 180 ccacccgctt ctggtgaaca tctttaccaa caatacattg cgaccaaagc agaaaccaat 240 attaatcagt tacgtgcgaa tgttgaggta cttcttggtg aaattagcag ttcaatgagt 300 gatacgctca gtgacaccag ttcctttgct aatgtgattg ataaaagctt taaggattta 360 gagcgcgtcg agcaagacaa tctctcgatt gaagaagtaa tgacggtgat ccgccgcttg 420 gtgagtgact ctaaagatat tcgacactca accaatttcc taaataatca actgaacgcg 480 gcaacactag aaatctctcg tcttaaagag cagctggcga aagttcagaa agatgctctg 540 tttgacagtt tatctggact ctataaccgc cgagcttttg atggcgatat gttcacgctg 600 atccatgcag gtcaacaagt cagcctgatc atgctcgaca tcgaccactt caaagccctt 660 aatgataact atggccacct gtttggtgac caaattatcc gtgcgatcgc caaacgtctt 720 caaagcctat gccgtgacgg cgtgacagct tatcgttatg gcggtgaaga gtttgcactg 780 attgctccgc acaaatcgct gcgtattgca cgccagtttg ctgaatcggt gcgacgttca 840 atagaaaagc tcaccgtaaa agatcggcgt agcggtcaat cggtcggtag cattaccgct 900 tcgtttggtg tagtagaaaa gattgaaggt gactctttgg agtctcttat cggtcgagcg 960 gatggattgc tgtatgaagc gaaaaatctg ggccgcaatc gagtcatgcc gctctaa 1017 <210> 48 <211> 338 <212> PRT
<213> Vibrio cholerae <400> 48 Met Thr Thr Glu Asp Phe Lys Lys Ser Thr Ala Asn Leu Lys Lys Val Val Pro Leu Met Met Lys His His Val Ala Ala Thr Pro Val Asn Tyr Ala Leu Trp Tyr Thr Tyr val Asp Gin Ala Ile Pro Gin Leu Asn Ala r r r Glu Met Asp Ser Val Leu Lys Asn Phe Gly Leu Cys Pro Pro Ala Ser Gly Glu His Leu Tyr Gln Gln Tyr Ile Ala Thr Lys Ala Glu Thr Asn Ile Asn Gln Leu Arg Ala Asn Val Glu Val Leu Leu Gly Glu Ile Ser Ser Ser Met Ser Asp Thr Leu Ser Asp Thr Ser Ser Phe Ala Asn Val Ile Asp Lys Ser Phe Lys Asp Leu Glu Arg Val Glu Gln AS Asn Leu Ser Ile Glu Glu Val Met Thr Val Ile Arg Arg Leu Val Ser Asp Ser Lys Asp Ile Arg His Ser Thr Asn Phe Leu Asn Asn Gln Leu Asn Ala Ala Thr Leu Glu Ile Ser Arg Leu Lys Glu Gln Leu Ala Lys Val Gln Lys Asp Ala Leu Phe Asp Ser Leu Ser Gly Leu Tyr Asn Arg Arg Ala Phe ASp Gly Asp Met Phe Thr Leu Ile His Ala Gly Gln Gln Val Ser Leu Ile Met Leu Asp Ile Asp His Phe Lys Ala Leu Asn Asp Asn Tyr Gly His Leu Phe Gly Asp Gln Ile Ile Arg Ala Ile Ala Lys Arg Leu Gln Ser Leu Cys Arg Asp Gly Val Thr Ala Tyr Arg Tyr Gly Gly Glu Glu Phe Ala Leu Ile Ala Pro His Lys Ser Leu Arg Ile Ala Arg Gln Phe Ala Glu Ser Val Arg Arg Ser Ile Glu Lys Leu Thr Val Lys Asp Arg Arg Ser Gly Gln Ser Val Gly Ser Ile Thr Ala Ser Phe Gly val Val Glu Lys Ile Glu Gly Asp Ser Leu Glu Ser Leu Ile Gly Arg Ala Asp Gly Leu Leu Tyr Glu Ala Lys Asn Leu Gly Arg Asn Arg Val met Pro Leu <210> 49 <211> 1374 <212> DNA
<213> vibrio cholerae <400> 49 tcaaaagcga tagagtgggt tttgcctacg cttagcggta tacatacgtt catcggccag 60 tttgaacatt tcatcaggtg tggcaaacga ctggtcatac aaagcatatc cgatacttac 120 acgaacatgg ataagcttgt cgtcataaac gatgggcgtt tcagaaatcc tttttaaaat 180 attgtcactg actttaagca cgtcttgttc acgatgaatt cgtggaatta acacgagaaa 240 ctcatccccc ccaatccgcg ccaccagatc ggaaacccgc aggctcgatt taattctttc 300 cgcacaagcc accagcactt tatcgcctgc gctatgtcca tgggaatcgt tgatagattt 360 aaaacggtca atatcaatgt tcaacaaagc aaagttacct tcgctatgag agcgcttagc 420 attttcaaag tagtgttcaa tggtatagat aaaatagcgc cgattcggca agtgggttaa 480 agggtcatgt agcgcacgct cctccgcgac ttgataaagg cgcatgataa cgccaaagcc 540 tgccatcaat accaataaca ccgagtatcc caacaagcgc actgcatttc gggtatacca 600 agataactgc tgtagtaaat cttgcttttc agcgaccgca attcgccaac ttccgtaagg 660 gaaatagaca ttctcttgtg caaaagcgtg ctcaaatact cgaggctctc caaaaaacac 720 gtccccctca ctgccacggc tgtctaaacc acgaatcgca acctgaaaat gctccccaaa 780 gctgtaaata ctggttgctg aaagcaatga atcccaatcc atcaccacac tcagtacccc 840 ccaataacgc gtatccttcg gtgggtcgta gaatatcggt tctcgaatca ccagcgcgcg 900 cccaccttga acgagatcga caggtccaga gacgaacgtc tgtttgattt cacgtgcttt 960 ttttattgac tgccactgct gaggaacggt gcggtaatcc aaaccgagta gtgcattggt 1020 ttgaggaagc ggatagctga aagcgaccac atcattaggg gcgataccta atgagcgtaa 1080 gtgatcgcta ttcctgatca ccgccgctga aagcggctcc cattgataga tattgaggtc 1140 gggatctagg gttaacaggg ttgttaaacc ttttacggta tagatatcac ccaaaatctc 1200 agcttctaat tgaaaacgta cgatggaaag atcttcttta gcttgttgac gtaaaccctc 1260 ttgtagatca cgtgtatggc taatatgaag ggattcaata accgcaatgc ccaaaaagag 1320 taaggcgaga aaataaattg agacatactt atatttgtgc gaggttaacc ccat 1374 <210> 50 <211> 457 <212> PRT
<213> vibrio cholerae <400> 50 Met Gly Leu Thr Ser His Lys Tyr Lys Tyr Val Ser Ile Tyr Phe Leu Ala Leu Leu Phe Leu Gly Ile Ala Val Ile Glu Ser Leu His Ile Ser His Thr Arg Asp Leu Gln Glu Gly Leu Arg Gin Gin Ala Lys Glu Asp Leu Ser Ile Val Arg Phe Gin Leu Glu Ala Glu Ile Leu Gly Asp Ile Tyr Thr Val Lys Gly Leu Thr Thr Leu Leu Thr Leu Asp Pro Asp Leu Asn Ile Tyr Gin Trp Glu Pro Leu Ser Ala Ala Val Ile Arg Asn Ser Asp His Leu Arg Ser Leu Gly Ile Ala Pro Asn Asp Val Val Ala Phe Ser Tyr Pro Leu Pro Gin Thr Asn Ala Leu Leu Gly Leu Asp Tyr Arg Thr Val Pro Gin Gin Trp Gin Ser Ile Lys Lys Ala Arg Glu Ile Lys Gin Thr Phe Val Ser Gly Pro Val Asp Leu Val Gin Gly Gly Arg Ala Leu Val Ile Arg Glu Pro Ile Phe Tyr Asp Pro Pro Lys Asp Thr Arg Tyr Trp Gly Val Leu Ser val Val Met Asp Trp Asp Ser Leu Leu Ser Ala Thr Ser Ile Tyr Ser Phe Gly Glu His Phe Gin Val Ala Ile Arg Gly Leu Asp Ser Arg Gly Ser Glu Gly Asp Val Phe Phe Gly Glu Pro Arg Val Phe Glu His Ala Phe Ala Gin Glu Asn Val Tyr Phe Pro Tyr Gly Ser Trp Arg Ile Ala Val Ala Glu Lys Gin Asp Leu Leu Gin Gin Leu Ser Trp Tyr Thr Arg Asn Ala Val Arg Leu Leu Gly Tyr Ser Val Leu Leu Val Leu Met Ala Gly Phe Gly Val Ile Met Arg Leu Tyr Gin Val Ala Glu Glu Arg Ala Leu His Asp Pro Leu Thr His Leu Pro Asn Arg Arg Tyr Phe Ile Tyr Thr Ile Glu His Tyr Phe Glu Asn Ala Lys Arg Ser His Ser Glu Gly Asn Phe Ala Leu Leu Asn Ile Asp Ile Asp Arg Phe Lys Ser Ile Asn Asp Ser His Gly His Ser Ala Gly Asp Lys Val Leu Val Ala Cys Ala Glu Arg Ile Lys Ser Ser Leu Arg Val Ser Asp Leu Val Ala Arg Ile Gly Gly Asp Glu Phe Leu Val Leu Ile Pro Arg Ile His Arg Glu Gin Asp Val Leu Lys Val Ser Asp Asn Ile Leu Lys Arg Ile Ser Glu Thr Pro Ile Val Tyr Asp Asp Lys Leu Ile His Val Arg Val Ser Ile Gly Tyr Ala Leu Tyr Asp Gin Ser Phe Ala Thr Pro Asp Glu Met Phe Lys Leu Ala Asp Glu Arg Met Tyr Thr Ala Lys Arg Arg Gin Asn Pro Leu Tyr Arg Phe <210> 51 <211> 1176 <212> DNA
<213> Vibrio cholerae <400> 51 atggatagct ttgctggcaa ccaattaaaa gagatgacag agatgcgttt tgctcgtaag 60 cagcatattg tcctgatcag ctctggtgtt gctaccgcta tttttcttgg gtttgccctt 120 tactactatt ttaaccatca acccctgtca tccggtttat tgttattaag cggtattgtc 180 accttattga atatgatttc gctgaatcgt caccgcgaat tacacactca agccgattta 240 attctgtcat taattctgct cacttatgcg ctggccttag tcagcaatgc tcagcatgaa 300 ttatcgcatc tcttatggtt atatccgctc atcaccactt tagtcatgat taaccctttt 360 cggttaggct tggtttacag tgcagcgata tgcttagcga tgaccgcctc tatccttttt 420 aatccggcac aaactggctc gtaccctatt gcacagacct attttttagt aagtctattt 480 acgctgacga ttatctgtaa taccgcttct ttctttttct caaaagcgat caattatatt 540 cataccctat accaagaagg tattgaagag ttggcttatc ttgatccgtt aacgggctta 600 gccaatcgtt ggagctttga aacttgggcc acagaaaagc tcaaagaaca acagagttcg 660 aataccatta ccgcgcttgt ttttctggat attgataatt tcaaacgcat taatgacagt 720 tacggccatg atgttggcga tcaggtgtta aaacattttg cacaccgtct acgcaataat 780 attcgtaata aagatcgagc caccaatcaa catgattatt ccattgctcg atttgctggt 840 gatgagtttg tgctcttgtt atatggtgtg cgaaatttgc gtgatctcga taatattctc 900 aaccgtatct gtaatctctt cgtcgaccgc tatcctgaga cggatatgct caacaacctc 960 acggtgagta taggggcagc tatttatccc aaagatgcga tcactctgcc ggaactaacc 1020 cgctgcgcag ataaagccat gtatgccgct aaacacggtg gaaaaaatca gtaccgctat 1080 taccatgatg ccgctttccc tccggctgta gaaaccgtat taggcagtca gcccgttgag 1140 gctcctaacg taactccact gaaaaaagcg cactaa 1176 <210> 52 <211> 391 <212> PRT
<213> Vibrio cholerae <400> 52 Met Asp Ser Phe Ala Gly Asn Gin Leu Lys Glu Met Thr Glu Met Arg Phe Ala Arg Lys Gin His Ile Val Leu Ile Ser Ser Gly val Ala Thr Ala Ile Phe Leu Gly Phe Ala Leu Tyr Tyr Tyr Phe Asn His Gin Pro Leu Ser Ser Gly Leu Leu Leu Leu Ser Gly Ile Val Thr Leu Leu Asn Met Ile Ser Leu Asn Arg His Arg Glu Leu HIS Thr Gin Ala Asp Leu Ile Leu Ser Leu Ile Leu Leu Thr Tyr Ala Leu Ala Leu Val Ser Asn Ala Gin His Glu Leu Ser His Leu Leu Trp Leu Tyr Pro Leu Ile Thr Thr Leu Val Met Ile Asn Pro Phe Arg Leu Gly Leu Val Tyr Ser Ala Ala Ile Cys Leu Ala Met Thr Ala Ser Ile Leu Phe Asn Pro Ala Gin , , Thr Gly Ser Tyr Pro Ile Ala Gin Thr Tyr Phe Leu val Ser Leu Phe Thr Leu Thr Ile Ile cys Asn Thr Ala Ser Phe Phe Phe Ser Lys Ala Ile Asn Tyr Ile His Thr Leu Tyr Gin Glu Gly Ile Glu Glu Leu Ala Tyr Leu Asp Pro Leu Thr Gly Leu Ala Asn Arg Trp Ser Phe Glu Thr Trp Ala Thr Glu Lys Leu Lys Glu Gin Gin Ser Ser Asn Thr Ile Thr Ala Leu Val Phe Leu Asp Ile Asp Asn Phe Lys Arg Ile Asn Asp Ser Tyr Gly His Asp Val Gly Asp Gin Val Leu Lys His Phe Ala His Arg Leu Arg Asn Asn Ile Arg Asn Lys Asp Arg Ala Thr Asn Gin His Asp Tyr Ser Ile Ala Arg Phe Ala Gly Asp Glu Phe Val Leu Leu Leu Tyr Gly Val Arg Asn Leu Arg Asp Leu Asp Asn Ile Leu Asn Arg Ile Cys Asn Leu Phe Val Asp Arg Tyr Pro Glu Thr Asp met Leu Asn Asn Leu Thr Val Ser Ile Gly Ala Ala Ile Tyr Pro Lys Asp Ala Ile Thr Leu Pro Glu Leu Thr Arg Cys Ala Asp Lys Ala Met Tyr Ala Ala Lys His Gly Gly Lys Asn Gin Tyr Arg Tyr Tyr His Asp Ala Ala Phe Pro Pro Ala Val Glu Thr val Leu Gly Ser Gin Pro val Glu Ala Pro Asn val Thr Pro Leu Lys LyS Ala His <210> 53 <211> 1911 <212> DNA

Pa6PP1.P611 611PD11D66 PPDDlaDa6P e6D1PDalle DEP6PD111D

111.1p6D1D6 PD1DP6PDPD PPP6a1666D ED1PD6D1PD DPDDPD1P66 6PlaDPP1PD

6D6DDPD6D6 DaDaPeDaDD P6aDDDPala PPP6PDADP P61P61P66a laPDDP31-DP
OtLT
111E1Daell u661DeppDa 6DPaPPEaDD 16DD116P6D PPD1PDP1DP 6DaDaD4PPD

DaaDPPa6DP 66DP1DPPDP 6p4666611.1 De66)6666E )61PpD6Dp1 6ED6Dep161 zlauaD6D6a DPDP1aPD6D DEP6aPPD16 Dall61066P P6PDDDaP6P 61414E361D

pl1DEEDD66 161D11131u D6eD6ppll DDPPPDAPD 1E661a6DD1 PalaD6P6DD

ppplpaplel 6rD66eppup 6D1PDPPD1P 636D1DPDD1 P6DDP61DPE 6366E6 OttT
laD6PDPPD1 ap6a646Dpe 611T616ael 6pplp6D11D leluDD6p1D 61pep1App pp611D3361 11611311pr DDPPPD66DP DP116D616D 66DP6PPP6P 51116D61D1 OZET
111.D166e6D 6DITDDllup ppuep66661 16PD6PDPPD ADDEIPPD3P 1D6Dpallpp 111661DP6D PP1PDDaPPD 66P6PP6Da6 66 616PD6PDPP PD66611D1P
OUT
pluppplp61 Dplpb16pD6 plap111D66 DeaDye61D6 u6lpplupp) lpve6D6epu OM
1D6E146361 pple661D61 DDD1611DaD 6DPPDPDDPD 66 D6PPaPPDEIP

u46616D661 aD6DaPalaP PDAPPP6D6 61p166app1. DD1P6aDD3P ullpDp6Dp6 OZOT
6161611)u) lepluD6u6v 6pD6616ple ppeD1D6r61 Deuu61p661 PDPEaD361P

D1pDle166D 116e6DEllp eop1D1p16D 1D6D1P1D1D lppe66EuD1 PDDPD1PDD1 DupuuullDD 6D1x66D661 Dep6D6D616 61pD661E61 66Dp6pe6lp 1)666pp6E6 Ot8 pp61x11136 p6puuDappl luu61D6upp plplluDD6e 16166166D1 eD66D6ppu6 lpap1D661) 6616P6a1PP Dp6e6luppl DP1PD6P6DD PPD6611D1P DaDall6PDD
OZL
Du61131ell D616Dep616 D6apepp6p3 6D66e6pupp D6PEDDAPP Plaa6D6PPP

6p6D1.66eDu u61D161p6u PaPPD1PD66 1p6p16P161 P111P6aDaP 66D1DEDuP

DDDADEODE 1DaDDEIP6154 6111PEDDPP DPP1D611.66 PD6E66aPPP PDPPDa6D1P
OVS
DaDPPDP1DP DDP6PPaDaD aD6D6PP611 laPDaaDD6D 16DD6111.61 e16611p6a6 08t P1101E6366 111116D6D1 el6e1116D lauullvD6e ep661D1Deu D1D6DE11D6 OZt 6)6166appa upp6r)16D1 uup6616pe6 6666 le66116616 eppep16116 16D66Dap61 PDD1PDDD6P 611161D1ru ela6uplapl 116611D66D D6a6lpelur 00E DDDPPDDaPa DDDP1PD1D6 D6aDDaPPP6 6DP166DP61 u631plalla u61.D16Dul6 OtZ ple6D1Dell 6plETD661P 611.161:D636 1PpplEpl6e DlE0P6P166D 611PDDPDu6 081 upu6e6p66 1D1p11)D6) 6e111D6611 1666166Del PPP1PDDPDP PDPPDa6PP6 OZT 1D1D6DueDu PDPPDPP6aa Da6PDPPD6D 13PDDPDPPD 1.1vu6D1r1D p111.1166D1 09 lze6116116 aplle11161 66D611p6le 666PD6Da6P aDPPDPPPDP 1r1D6De6le ES <00t>
apJaLop opw <ETZ>

, .
<210> 54 <211> 636 <212> PRT
<213> vibrio cholerae <400> 54 Met Thr Leu Tyr Lys Gin Leu Val Ala Gly met Ile Ala Val Phe Ile Leu Leu Leu Ile Ser Val Phe Thr Ile Glu Phe Asn Thr Thr Arg Asn Ser Leu Glu Gin Gin Gin Arg Ser Glu Val Asn Asn Thr Ile Asn Thr val Gly Leu Ala Leu Ala Pro Tyr Leu Glu Lys Lys Asp Thr Ile Ala Val Glu Ser Val Ile Asn Ala Leu Phe Asp Gly Ser Ser Tyr Ser Ile val Arg Leu Ile Phe Leu Asp Asp Gly Thr Glu Ile Leu Arg Ser Tyr Pro Ile Gin Pro Asn Asn Val Pro Ala Trp Phe Thr Gin Leu Asn Leu Phe Glu Pro Ile His Asp Arg Arg val Val Thr Ser Gly Trp met Gin Leu Ala Glu val Glu Ile Val Ser His Pro Gly Ala Ala Tyr Ala Gin Leu Trp Lys Ala Leu Ile Arg Leu Ser Ile Ala Phe Leu Ala Ile Leu Val Ile Gly met Phe Ala val Ala Phe Ile Leu Lys Arg Ser Leu Arg Pro Leu Gin Leu Ile val Asn Lys met Glu Gin val Ala Asn Asn Gin Phe Gly Glu Pro Leu Pro Arg Pro Asn Thr Arg Asp Leu Ile Tyr val val Asp Gly Ile Asn Lys Met Ser Glu Gin Val Glu Lys Ala Phe Lys Ala Gin Ala Lys Glu Ala Gin Gin Leu Arg Glu Arg Ala Tyr Leu Asp Pro Val Ser His Leu Gly Asn Arg Ala Tyr Tyr met Ser Gin Leu Ser Gly Trp Leu Ser Glu Ser Gly Ile Gly Gly Val Ala Ile Leu Gln Ala Glu Phe Ile Lys Glu Leu Tyr Glu Glu Lys Gly Tyr Glu Ala Gly Asp Gly Met Val Arg Glu Leu Ala Asp Arg Leu Lys Asn Ser Ile Thr Ile Lys Asp Ile Ser Ile Ala Arg Ile Ser Thr Tyr Glu Phe Gly Ile Ile Met Pro Asn Met Asp Glu Thr Glu Leu Lys Ile Val Ala Glu Ser Ile Ile Thr Cys Val Asp Asp Ile Asn Pro Asp Pro Thr Gly Met Ala Lys Ala Asn Leu Ser Leu Gly Val Val Ser Asn Lys Arg Gln Ser Ser Thr Thr Thr Leu Leu Ser Leu Leu Asp Asn Ala Leu Ala Lys Ala Lys Ser Asn Pro Glu Leu Asn Tyr Gly Phe Ile Ser Ser Asp Thr Asp Lys Ile Ile Leu Gly Lys Gln Gln Trp Lys Thr Leu Val Glu Glu Ala Ile His Asn Asp Trp Phe Thr Phe Arg Tyr Gln Ala Ala Asn Ser Ser Trp Gly Lys Thr Phe His Arg Glu Val Phe Ser Ala Phe Glu Lys Asp Gly Val Arg Tyr Thr Ala Asn Gln Phe Leu Phe Ala Leu Glu Gln Leu Asn Ala Ser His Ile Phe Asp Gin Tyr Val Ile Glu Arg Val Ile Gln Gln Leu Glu Lys Gly Glu Leu Thr Asp Pro Leu Ala Ile Asn Ile Ala Gln Gly Ser Ile Ser Gln Pro Ser Phe Ile Arg Trp Ile Ser Gln Thr Leu Ser Lys His Leu Ser val Ala Asn Leu Leu His Phe Glu Ile Pro Glu Gly cys Phe val Asn Glu Pro His Tyr Thr Ala Leu Phe Cys Asn Ala Val Arg Asn Ala Gly Ala Asp Phe Gly Val Asp Asn Tyr Gly Arg Asn Phe Gln Ser Leu Asp Tyr Ile Asn Glu Phe Arg Pro Lys Tyr Val Lys Leu Asp Tyr Leu Phe Thr His His Leu Asp Asp Glu Arg Gin Lys Phe Thr Leu Thr Ser Ile Ser Arg Thr Ala His Asn Leu Gly Ile Thr Thr Ile Ala Ser Arg Val Glu Thr Gln Thr Gln Leu Asp Phe Leu Ser Glu His Phe Ile Glu Val Phe Gln Gly Phe Ile Val Asp Lys <210> 55 <211> 1581 <212> DNA
<213> Vibrio cholerae <400> 55 ctactcaaca cacacttggt tacggccatt ggctttggcg cgatacaaag ctttgtcagc 60 gcggtagaac gtacgttggg tattttcccc ctcgcgatgc aaggtgatac cgatactgac 120 cgtcagtccc cgttcgccaa gtacgtcttg ccatgggaaa tcaaaaatac gttggcgata 180 ggtttcggca tgcatttgtg ccatatcact ggtgacgttt tccaaaatca ccagaaattc 240 ctcgccaccg aaacgtacgc aggaggcacc acggaattta aagtaactcg ccagttcact 300 ggatacattg acaatcgctt tatcccctac caaatgactc aattcatcat tgatcgattt 360 aaagtggtca atatcaacga ctaagaaagc aaacggggtt tcgtgcagca gcagatcttt 420 cagcttcacg tccaaccaac ggcggttatg cagttttgtc agtggatcgg tgaacacatc 480 ttgctgtagt tgcaacaccg tattcttctg gctttcggtg gtttctttta gctcacgatt 540 ttctaattcc gacaaaatca gtttaagttg tagctcaaag cgcgataggc ggcgtagctg 600 aattgggcct aattcactga tggggatccg cttcatcaaa tcgctttcga tgcgaaatgc 660 tttcttttcg taaaccagtg cggttttgta cattccttcg agttcacaca cttcgctgaa 720 cgcttcatag aggcgttttt caaggaaagg ggaatgaatg ttttgtaagc gcttttcagt 780 gctacccagc agcatggtgg caaaatgcgc cttacctgct ttagagaggc aatgcgctaa 840 ctcgatgcgt agcatgcttg atagccaatc cgatggcgtc agcgatgacg aatactgtgc 900 attggcgagt gtcatcatcg ccttttgcac tttgccttgt tgcagataaa gcttggcttg 960 atagagcatg atctgcccag tcagcagttt atcgctgacc agaatgctca actcatcaca 1020 ctcttttatc agatcattgg ccgctgcata acgaccaagg ctgatgtagc aagccagcat 1080 atacagcttg taacgcaggc gcagtgagcg gctagaaatc gcatgatcta tgctgtcaat 1140 tttttggtag tagcgtaacg cacggctgtg atcgccataa gcatcacata aattgcccat 1200 tccgagcact gcaagtacgt agtcatcaat catgccatgc tcaacggcga tgttggatat 1260 cgcaacgtat tcagacagtg ccgcgacata ttcaccatgg tcgagtaaac gctcactcaa 1320 actgtgtttg accgagagca ttaattccag atccgtcggt aactctaata gggaaagagc 1380 ggcgcgcagc tcttcaatac tggtttgcca ctgtttcatt tcgcggcggt attcggcgct 1440 gatgatgtag ctttgtgcac gctcttgggc ggtggttgcc acgtgctgtc tgacatggtt 1500 ccagaaaatg atcgcctctt caccagcgac agcggccgca tccagtcccg cttctttgat 1560 cttattgagc agggtttcca t 1581 <210> 56 <211> 526 <212> PRT
<213> Vibrio cholerae <400> 56 Met Glu Thr Leu Leu Asn Lys Ile Lys Glu Ala Gly Leu Asp Ala Ala Ala Val Ala Gly Glu Glu Ala Ile Ile Phe Trp Asn His Val Arg Gin His Val Ala Thr Thr Ala Gin Glu Arg Ala Gin Ser Tyr Ile Ile Ser Ala Glu Tyr Arg Arg Glu Met Lys Gin Trp Gin Thr Ser Ile Glu Glu Leu Arg Ala Ala Leu Ser Leu Leu Glu Leu Pro Thr Asp Leu Glu Leu Met Leu Ser val Lys His Ser Leu Ser Glu Arg Leu Leu Asp His Gly Glu Tyr Val Ala Ala Leu Ser Glu Tyr val Ala Ile Ser Asn Ile Ala Val Glu His Gly Met Ile Asp Asp Tyr Val Leu Ala Val Leu Gly Met Gly Asn Leu Cys Asp Ala Tyr Gly Asp His Ser Arg Ala Leu Arg Tyr Tyr Gin Lys Ile Asp Ser Ile Asp His Ala Ile Ser Ser Arg Ser Leu Arg Leu Arg Tyr Lys Leu Tyr Met Leu Ala Cys Tyr Ile Ser Leu Gly Arg Tyr Ala Ala Ala Asn Asp Leu Ile Lys Glu Cys Asp Glu Leu Ser Ile Leu Val Ser Asp Lys Leu Leu Thr Gly Gln Ile Met Leu Tyr Gln Ala Lys Leu Tyr Leu Gln Gln Gly Lys Val Gln Lys Ala Met Met Thr Leu Ala Asn Ala Gln Tyr Ser Ser Ser Leu Thr Pro Ser Asp Trp Leu Ser Ser Met Leu Arg Ile Glu Leu Ala His Cys Leu Ser Lys Ala Gly Lys Ala His Phe Ala Thr Met Leu Leu Gly Ser Thr Glu Lys Arg Leu Gln Asn Ile His Ser Pro Phe Leu Glu Lys Arg Leu Tyr Glu Ala Phe Ser Glu Val Cys Glu Leu Glu Gly Met Tyr Lys Thr Ala Leu Val Tyr Glu Lys Lys Ala Phe Arg Ile Glu Ser Asp Leu Met Lys Arg Ile Pro Ile Ser Glu Leu Gly Pro Ile Gln Leu Arg Arg Leu Ser Arg Phe Glu Leu Gln Leu Lys Leu Ile Leu Ser Glu Leu Glu Asn Arg Glu Leu Lys Glu Thr Thr Glu Ser Gln Lys Asn Thr Val Leu Gln Leu Gln Gln Asp Val Phe Thr Asp Pro Leu Thr Lys Leu His Asn Arg Arg Trp Leu Asp Val Lys Leu Lys Asp Leu Leu Leu His Glu Thr Pro Phe Ala Phe Leu Val Val Asp Ile Asp His Phe Lys Ser Ile Asn Asp Glu Leu Ser His Leu Val Gly Asp Lys Ala Ile Val Asn Val Ser Ser Glu Leu Ala Ser Tyr Phe Lys Phe Arg Gly Ala Ser Cys Val Arg Phe Gly Gly Glu Glu Phe Leu Val Ile Leu Glu Asn Val Thr Ser Asp met Ala Gin Met His Ala Glu Thr Tyr Arg Gin Arg Ile Phe Asp Phe Pro Trp Gin Asp val Leu Gly Glu Arg Gly Leu Thr Val Ser Ile Gly Ile Thr Leu His Arg Glu Gly Glu Asn Thr Gin Arg Thr Phe Tyr Arg Ala Asp Lys Ala Leu Tyr Arg Ala Lys Ala Asn Gly Arg Asn Gin Val Cys Val Glu <210> 57 <211> 1101 <212> DNA
<213> Vibrio cholerae <400> 57 tcacgatgag gggctttttt gtaggaattt catttcatac atgtttttat ctgccagatg 60 gatcaactgg ctcaaattgg tgctgtcgag tggataagta ctgaccccga cgctggtgtt 120 gagcttggct cgtaaatcgc cacttaattc aaattcatgg tcgaaacact gtttgatcat 180 gcgctgcatc atcatctgct cggtcgaatt gatgctgctt aggatgatgg caaattcatc 240 tccccccatc cgaaacacac gataatcgaa tgaaggaatc gagttgttta agcgataagc 300 aacctgtttg agtaccgcat cgcccatttg atggccgtag gtatcattaa tttgtttaaa 360 accattcaga tcgagcaaaa agagagagaa tccaccgctg cggcggtggc gttctaattc 420 ggcgaacatg gctgtgcggt tttccagccc tgttaatggg tccgttaagg ccaagactct 480 atggtgcgtg gcctctttat gcaaaataaa actcaccagt cccacacagc taaacgtcaa 540 caaaattaac gcaaactgga tgcgactgag gtaattcagt ttctcttttt gctctacata 600 caaaggactt tgcattccaa atgtgcggtt tatgaactga ataaaaatct ccagctcttg 660 ttgggcggca acaataaaag tttgtaagct ttctggattt ttggccgcaa gcagtagcgg 720 ttcaagttgt ttaaagcgcg caaacgcggc ttggaagaat tcgcgagtgc tgggcatgcc 780 tataatgccg tcggcttctg ggctattgag gatcagatca aaacggctcc aagtcagctc 840 atatttcacc atcacatcgc gctggttgct ctccgactcc aataggtagg gggagagtgc 900 cagcatctca gtaaactctt tattgagctg gaataagaac cagatcgctt ggttagtatg 960 cgaagagtaa gacttagata aatcgcgagt actgttgatc aaatacaaat tggccaaaat 1020 cagaatcgcc gacatgaaga tcagcagtgt tttggcatgt aagatcagcg ggtggagcgt 1080 tttctgagtt tgtgtattca t 1101 , <210> 58 <211> 366 <212> PRT
<213> vibrio cholerae <400> 58 Met Asn Thr Gin Thr Gin Lys Thr Leu His Pro Leu Ile Leu His Ala Lys Thr Leu Leu Ile Phe Met Ser Ala Ile Leu Ile Leu Ala Asn Leu Tyr Leu Ile Asn Ser Thr Arg Asp Leu Ser Lys Ser Tyr Ser Ser His Thr Asn Gin Ala Ile Trp Phe Leu Phe Gin Leu Asn Lys Glu Phe Thr Glu Met Leu Ala Leu Ser Pro Tyr Leu Leu Glu Ser Glu Ser Asn Gin Arg Asp Val Met Val Lys Tyr Glu Leu Thr Trp Ser Arg Phe Asp Leu Ile Leu Asn Ser Pro Glu Ala Asp Gly Ile Ile Gly Met Pro Ser Thr Arg Glu Phe Phe Gin Ala Ala Phe Ala Arg Phe Lys Gin Leu Glu Pro Leu Leu Leu Ala Ala Lys Asn Pro Glu Ser Leu Gin Thr Phe Ile val Ala Ala Gin Gin Glu Leu Glu Ile Phe Ile Gin Phe Ile Asn Arg Thr Phe Gly Met Gin Ser Pro Leu Tyr Val Glu Gin Lys Glu Lys Leu Asn Tyr Leu Ser Arg Ile Gin Phe Ala Leu Ile Leu Leu Thr Phe Ser cys Val Gly Leu Val Ser Phe Ile Leu His Lys Glu Ala Thr His His Arg Val Leu Ala Leu Thr Asp Pro Leu Thr Gly Leu Glu Asn Arg Thr Ala Met Phe Ala Glu Leu Glu Arg HiS Arg Arg Ser Gly Gly Phe Ser Leu a Phe Leu Leu Asp Leu Asn Gly Phe Lys Gin Ile Asn Asp Thr Tyr Gly His Gin Met Gly Asp Ala Val Leu Lys Gin val Ala Tyr Arg Leu Asn Asn Ser Ile Pro Ser Phe Asp Tyr Arg Val Phe Arg Met Gly Gly Asp Glu Phe Ala Ile Ile Leu Ser Ser Ile Asn Ser Thr Glu Gin Met met Met Gin Arg Met Ile Lys Gin Cys Phe Asp His Glu Phe Glu Leu Ser Gly Asp Leu Arg Ala Lys Leu Asn Thr Ser Val Gly Val Ser Thr Tyr Pro Leu Asp Ser Thr Asn Leu Ser Gin Leu Ile His Leu Ala Asp Lys Asn Met Tyr Glu Met Lys Phe Leu Gin Lys Ser Pro Ser Ser <210> 59 <211> 1824 <212> DNA
<213> Vibrio cholerae <400> 59 ttaggctaca ttcgtttctt ttctccagcg ttcaatcatc acactcggta aatcaggtcg 60 actgaagtaa tacccttgaa tttgctcaca gcccatttga tagagtttat ccagtgcttg 120 ttggttctct accccctcag cgacgagatc gagtttaagc tggttagcaa gctgaataat 180 caaccacacg atactctcag aggtttggtt ggtaagtagg ttacgcacaa atgcagcatc 240 aatcttgatg caatcaatcg gataactgtg aatgtagtta aggctcgaat aacctgtccc 300 aaaatcatcc aaggcaattt taaaacccaa ttcacgcaat atggtgagaa tactgcatac 360 ttctgcggcc ttagagagta aaaccgtttc tgtcagctca atagtgaact cgtcggcttg 420 aaaaccatag gctttaatgg tttttaatag atgctcaagg taacgattgg aatgcgtcag 480 ctcatcggcg gagcagttga tgcttaagcg aattttttgg tcaatacctt gttctaattc 540 ttgtttcgcg atgcaggcca attcgagaat acgttcgcca aattcgacaa tcaggccaga 600 ttgctctgct gcttcaatga attccaatgg cgttaccaca ccgagcgtac tgctattcca 660 acgcgttaag atctcaaaat agtcccaatt tctttgatgt tttttcacga tcggttgcac 720 gaccacatac agctcagttt gatggatagg cttactcaat tcactacgca gagcttcgat 780 gatttgtgta cgccgatagt attgattgct gagtaagttg tcgtagaaac gaatgcgtgt 840 gttatggttc cgtttacact cttttaaagc gagacttgca ttgaacagta attgatcggc 900 attgagcttt tcaccactgt atttggtaat accaatactg acactgattt tgagtcgacg 960 atcttgatcg atataatctt gcgccagctt gttgagtatg gtttggcaga tcttcatcgg 1020 ctcacgatct gtggttaaaa aagcaaattc atcagcggcg attcgaaagg cgtatccttc 1080 ttcggggacg gcttgtttta tcgcatccgc gacaaatttc agcacaagat ctcccaaata 1140 gtgcccatgc agatcgttta tcgaacgaaa ttcatcaata tcaagaaagg ccagagtgaa 1200 atgatgtcta tcttcttgaa cgagagccgt cagtttctcg gctaaatcat tacgattcat 1260 taaacccgtt aagttgtcgt gagatatttc atgacgtaat tgattgatta ggctctgaga 1320 gcgtacctcc atctgtttac attccagatc atgagcgatc atctgagcca aaatctggtg 1380 aactaacacg agattagcaa agtcgtctaa ctgacgcgta aaagtcgaga tcaaaacgcc 1440 gtagttttcg ccatttgaaa aataaatcgg gatacccaga tacgcctcaa tatggttctc 1500 aactaaataa gcatcgttag gaaaaagttc cgcgactttg cttgcaaata ggcaataagg 1560 ttgtctttgt aatccgactt gctcacaagg tgtgccttgt agttcgtaat acagctctaa 1620 actgctgggt tcgacactgg cacaacttaa gttatgagct ttgtagcgca ttttatctag 1680 ctcaatgacc attgagctgt ggctattgaa ggtgcggtgg agaaactgag tgatttgtga 1740 gagcaactcc aaccccccca gctgactgaa gtgatgtatg gaatctaggc tcagtttttc 1800 tgttatcagt tgagtcttgg tcat 1824 <210> 60 <211> 607 <212> PRT
<213> Vibrio cholerae <400> 60 Met Thr Lys Thr Gin Leu Ile Thr Glu Lys Leu Ser Leu Asp Ser Ile His His Phe Ser Gin Leu Gly Gly Leu Glu Leu Leu Ser Gin Ile Thr Gin Phe Leu His Arg Thr Phe Asn Ser His Ser Ser Met Val Ile Glu Leu Asp Lys Met Arg Tyr Lys Ala His Asn Leu Ser Cys Ala Ser Val Glu Pro Ser Ser Leu Glu Leu Tyr Tyr Glu Leu Gin Gly Thr Pro Cys Glu Gin Val Gly Leu Gin Arg Gin Pro Tyr Cys Leu Phe Ala Ser Lys Val Ala Glu Leu Phe Pro Asn Asp Ala Tyr Leu Val Glu Asn His Ile Glu Ala Tyr Leu Gly Ile Pro Ile Tyr Phe Ser Asn Gly Glu Asn Tyr , , ' 115 120 125 Gly Val Leu Ile Ser Thr Phe Thr Arg Gin Leu Asp Asp Phe Ala Asn Leu Val Leu Val His Gin Ile Leu Ala Gin Met Ile Ala His Asp Leu Glu Cys Lys Gin met Glu Val Arg Ser Gin Ser Leu Ile Asn Gin Leu Arg His Glu Ile Ser His Asp Asn Leu Thr Gly Leu Met Asn Arg Asn Asp Leu Ala Glu Lys Leu Thr Ala Leu Val Gin Glu Asp Arg His His Phe Thr Leu Ala Phe Leu Asp Ile Asp Glu Phe Arg Ser Ile Asn Asp Leu His Gly His Tyr Leu Gly Asp Leu Val Leu Lys Phe Val Ala AS

Ala Ile Lys Gin Ala Val Pro Glu Glu Gly Tyr Ala Phe Arg Ile Ala Ala Asp Glu Phe Ala Phe Leu Thr Thr Asp Arg Glu Pro met Lys Ile Cys Gin Thr Ile Leu Asn Lys Leu Ala Gin Asp Tyr Ile Asp Gin Asp Arg Arg Leu Lys Ile Ser Val Ser Ile Gly Ile Thr Lys Tyr Ser Gly Glu Lys Leu Asn Ala Asp Gin Leu Leu Phe Asn Ala Ser Leu Ala Leu Lys Glu Cys Lys Arg Asn His Asn Thr Arg Ile Arg Phe Tyr Asp Asn Leu Leu Ser Asn Gin Tyr Tyr Arg Arg Thr Gin Ile Ile Glu Ala Leu Arg Ser Glu Leu Ser Lys Pro Ile His Gin Thr Glu Leu Tyr Val val Val Gin Pro Ile Val Lys Lys His Gin Arg Asn Trp Asp Tyr Phe Glu Ile Leu Thr Arg Trp Asn Ser Ser Thr Leu Gly val val Thr Pro Leu , .

Glu Phe Ile Glu Ala Ala Glu Gin Ser Gly Leu Ile Val Glu Phe Gly Glu Arg Ile Leu Glu Leu Ala Cys Ile Ala Lys Gin Glu Leu Glu Gin Gly Ile Asp Gin Lys Ile Arg Leu Ser Ile Asn Cys Ser Ala Asp Glu Leu Thr His Ser Asn Arg Tyr Leu Glu His Leu Leu Lys Thr Ile Lys Ala Tyr Gly Phe Gin Ala Asp Glu Phe Thr Ile Glu Leu Thr Glu Thr val Leu Leu Ser Lys Ala Ala Glu Val Cys Ser Ile Leu Thr Ile Leu Arg Glu Leu Gly Phe Lys Ile Ala Leu Asp Asp Phe Gly Thr Gly Tyr Ser Ser Leu Asn Tyr Ile His Ser Tyr Pro Ile Asp Cys Ile Lys Ile Asp Ala Ala Phe Val Arg Asn Leu Leu Thr Asn Gin Thr Ser Glu Ser Ile Val Trp Leu Ile Ile Gin Leu Ala Asn Gin Leu Lys Leu Asp Leu Val Ala Glu Gly Val Glu Asn Gin Gin Ala Leu Asp Lys Leu Tyr Gin Met Gly Cys Glu Gin Ile Gin Gly Tyr Tyr Phe Ser Arg Pro As Leu Pro Ser Val Met Ile Glu Arg Trp Arg Lys Glu Thr Asn val Ala <210> 61 <211> 1998 <212> DNA
<213> Vibrio cholerae <400> 61 gtggcaggtc acaccttact ctcttccaac acgtttacgc cgctagaagc gtatcctgaa 60 gccttttggg catgggctgc gcagtttgat acttccgatg gtttgatccc ttttgccatc 120 aatacctgtc gctggaacta tttgccagtg atgggcggtg agtcgtttat ttttatgctg 180 gataatcatc ctcagcatcg gacttatctg atcattcaag cggcatgcgt cgataaagta 240 Z9 <00V>
aeJaLop opqo <ETZ>
flJd <ZTZ>
S99 <TTZ>
Z9 <OTZ>

6E161uEE 6E366E3E1E
0861 3613u631E1 EEEE611611 E11636611E 1166E3lEe6 63E66Eu336 EEEP636E6E
OZ61 3E1.611336E Eulu6636ee 6E361.E131.D 6E31P611aP P311e6u6u6 ADDPUelel 0981 4631314361 364E146614 E634p1633E 61.3E136E61 6616E3331E PPP6D613ED
0081 EE641e361e p6r36EE611 ElbuEEE163 11PlEuEu61 36613E113P 314E6E66E6 OVLT 33636EE631 eu336316E1 1113613111 6E6EE66661 6611E16316 1311613PE6 089T lED6E6431E ED6361.E3E6 316E31331E lEe16611E6 6361E61313 611E1E6366 0Z91 eE61131.E31 664E13623r e3EE311113 6663;11E33 E611E1E621361.431E616 09S1 3661ealEpp 1661.66E6pp PD1P3DP6PD PPE0611DeR E36E6613up 33E36E6143 00ST 6aE1633631 uu666u1116 313E613633 zP6E14PEP6 3661PEllae 6313EuuuE6 OtVT DPP3366ETP P35111P616 313PD631PE 1.6D6166P61 1D6U66136D PPE61PPD1D
08ET E1663131E6 DETPETED3) 6UPP3D6P6D 61:611316P P633PD1D3P EE31136111 OZET 61E16314E6 16Eu616361. 3PP3U)1.13e 61.13333311 63E6331E61 lleele6636 09Z1 63614E1413 361aE63333 E366EEE3E6 33E31361E3 3E61E63663 e6611661E1 00ZT 16631614E2 PE161DPEIDP 31.P131PDDD 11616P6P6P PP11161661 EIPP5DPPPD3 OKI DE1616uEED 361133E631 611P3P61PP PPEolnEoPP D1166163D1. 11E4331PE6 080T 1E631611E1 Eu366EE31.1 ElE663E666 66163E6666 E311E1E1.13 1361136EE6 OZOT 1E663E6111 1161663661 E3632E611E 66661EE633 upj53511 133316E36 096 631pEE1.646 46pu616E31 6E3e6aE161 36313161.3E E631661E36 E3361e3613 006 61E1.63E163 Euuu3Eu66u p6euEE163 11PlulaleE n63631.361 1E31366111 OV8 6316E64136 le6E36Euue 31E16E633E 11E6336E36 631EED1E1E 13PPDP631P
08L 33611E6613 66E1.E33E3E 61uE1661E6 16361.134E6 1363.E3361E E661366en OZL P66616E166 upE6631633 e3E1614433 66666 1661661E62 113E3616E6 099 EE61Ppople 11.436uE631. DaPlEPD4DD 6P6Ea4PPDD PA4DaP61P lEreDDDPDED
009 666. au63ElEE16 63E36E61E6 33E1661E11 3E16636E31 13611636Pu OVS ere6zEle61 1361161131 3636331E63 1E61146663 1.31631E3E6 361E1366E6 08V D166DIT6D 63DP3U1D6P 61X61.1D1PP DADD3DPED 316116E631 3636316En OZV 1E361661.36 PP61PDBEDP 61.1E1416E3 6E6E636E31 6314EEE616 E1133E16e3 09E 6366E6163e 1.14E61.631u eurElaa6E6 EEE631E36E E611rEu663 6E63E11361 00E PE3661.EuE6 36E3611Eul 16E3E141.11 1E66a16E61. 66331Eu313 PAPEaDDED

Met Ala Gly His Thr Leu Leu Ser Ser Asn Thr Phe Thr Pro Leu Glu Ala Tyr Pro Glu Ala Phe Trp Ala Trp Ala Ala Gln Phe Asp Thr Ser Asp Gly Leu Ile Pro Phe Ala Ile Asn Thr Cys Arg Trp Asn Tyr Leu Pro val Met Gly Gly Glu Ser Phe Ile Phe Met Leu Asp Asn His Pro Gln His Arg Thr Tyr Leu Ile Ile Gin Ala Ala Cys val Asp Lys val His Leu Ser Thr Gln Ser Gly Glu Leu Asp Phe Leu Gln Leu Ile Ala Ala Lys Trp Gln Cys Leu Arg Ala Glu Ile Glu Ala Ser Lys Glu Phe Lys Asn Arg Asp Leu Arg Glu Ala Gln Tyr Leu Ser Glu Ile Arg Gln Arg Glu Gln Phe Ile Asp Asn Met Lys Leu val His Gln Val Ala Leu Glu Leu Ser Asn Pro Ala Asn Leu Asp Glu Leu His Arg Ala Ser Val Glu Ala Met Arg His Arg Leu Gly Phe Asp Arg Ser Ala Leu Leu Leu Leu Asp Met Lys Lys Arg Cys Phe Ser Gly Thr Tyr Gly Thr Asp Glu His Gly Asn Thr Ile Asp Glu Gln His Thr Gln Tyr Asp Leu His Gln Leu Glu Pro Gln Tyr Leu Glu Ala Leu Ser Asn Glu Glu Cys Thr Leu Met val Val Glu Asp Val Pro Leu Tyr Thr Val Gly Gln Val val Gly Gln Gly Trp Asn Ala Met Leu Ile Leu Arg Asp Gly Asn Asp Thr Ile Gly Trp Ile Ala Ile Asp Asn Tyr Ile Asn Arg Gln Pro Ile Thr Glu Leu met Arg Ile Gly Asn Ile Leu Ser Arg His Ala Gin Ser Glu His Glu Leu Phe cys Arg Ile Gly Gly Glu Glu Phe Leu Leu Leu val Ala Asn Arg Ser Ala Glu Glu Ile His Leu Leu Ala Glu Asn Ile Arg Lys Ser Ile Glu Ala Glu cys Ile Glu His Cys Glu Asn Pro Ser Gly Glu Leu Leu Thr val Ser Ile Gly Tyr Ala Ala Ser Arg Tyr Lys Pro Arg Glu Ile Gin Phe Asp Gin Leu Tyr Ala Glu Ala Asp Lys Ala Leu Tyr Arg Ala Lys Ser Gin Gly Arg Asn Gin val Ile Gly val Ile val Glu Asn Ile AS cys Ile Gin Ala Glu met <210> 63 <211> 447 <212> DNA
<213> vibrio cholerae <400> 63 atgctagcgt tacctgcgga gtttgagcaa ttccattgga tggtcgatat ggttcagaat 60 gtcgatatgg gattgattgt gattaaccga gactacaacg tgcaagtgtg gaatgggttt 120 atgacccatc atagcggtaa gcaagctcat gatgttattg gtaaatctct gttcgagatt 180 tttccagaga tccctgtgga gtggtttaag ttaaaaacca aaccggtgta cgatctgggt 240 tgccgtagtt ttattacttg gcagcagcgc ccttatttgt tccattgccg taatgtgcgc 300 ccagtgactc agcaagccaa atttatgtat caaaacgtca cgcttaaccc aatgcgtaca 360 ccgacaggcg cgataaattc actcttctta tccattcaag atgcaacaag tgaagccctt 420 gtttctcaac aagcttcttc tcaataa 447 <210> 64 <211> 148 <212> PRT
<213> vibrio cholerae <400> 64 Met Leu Ala Leu Pro Ala Glu Phe Glu Gin Phe His Trp met val Asp Met val Gin Asn val Asp met Gly Leu Ile val Ile Asn Arg Asp Tyr Asn val Gin val Trp Asn Gly Phe Met Thr His His Ser Gly Lys Gin Ala His Asp val Ile Gly Lys Ser Leu Phe Glu Ile Phe Pro Glu Ile Pro Val Glu Trp Phe Lys Leu Lys Thr Lys Pro Val Tyr Asp Leu Gly cys Arg Ser Phe Ile Thr Trp Gin Gin Arg Pro Tyr Leu Phe His Cys Arg Asn val Arg Pro val Thr Gin Gin Ala Lys Phe met Tyr Gin Asn Val Thr Leu Asn Pro Met Arg Thr Pro Thr Gly Ala Ile Asn Ser Leu Phe Leu Ser Ile Gin Asp Ala Thr Ser Glu Ala Leu Val Ser Gin Gin Ala Ser Ser Gin <210> 65 <211> 1080 <212> DNA
<213> Vibrio cholerae <400> 65 tcagcgatga ccatgagttg aacccaatag cgcatgacaa tggtcaccat tgagttcaat 60 gacatgctct tcatcgaagc tgacgcggtt tttccccatt tttttcgaat gatagagagc 120 ttggtctgcg cgtttgaacc actgctccgg atcatcggtg cgaagtgctt cggctaaacc 180 gacactgacg gtgactttgg catggtatgg gtagtgcgtt tgttgaatcc gacaaccaat 240 atgactcatc acgagtgtag cgtcggttaa cgacgtattt tcaaacagca gtaaaaattc 300 atcgccccct aatcgaaaca acagatctaa ctcacggcag tgagtattca ttatttcaac 360 aacttgggta atgactttat ctcctgtgtc gtgtccataa aggtcattaa cagatttgaa 420 gtgatcgata tcgatcacgg cgatcaccgc cgattcattg gcgagctggc ggtggcgaag 480 acattttttc aaaaaaccat ccagttgatg acgattcaat gtgcccgtta atgcatgacg 540 agtggaaaga taaaaaagct cagtgtgcag cttacggata gcatctacca ccacatacat 600 gatggcggca caagcgctga tcgcaaggct aaagcgcaag gtgacttcgg cggtttgatg 660 gggaattaaa acccatatgc tggctggaat gataatggtg atggtcaata agttatcttt 720 ctgggggagt agaaaagcaa tcgcaatgag cacgggaaat agccagtagc tggcgagggt 780 gccgaaaatg tgaatagcca tcaccacgat gactaccacc aatgccagtg gaagcctaaa 840 accccatggt gttttctttt gataatagat agccgtaatt tcaatgagga gcgtgcattg 900 gaatacgatg atcaacccgc caagaagaac gtagtcaatc agcaagtttt taacggcgag 960 tggaaagaaa accaaactag aaataaaacc aataaaaagc gacacccgac gttgatagta 1020 agtgttcagt aactctgaac cggtaaaagc aggagagtga gtcgattttg tcatcgtcat 1080 <210> 66 <211> 359 <212> PRT
<213> Vibrio cholerae <400> 66 Met Thr Met Thr Lys Ser Thr His Ser Pro Ala Phe Thr Gly Ser Glu Leu Leu Asn Thr Tyr Tyr Gin Arg Arg val Ser Leu Phe Ile Gly Phe Ile Ser Ser Leu Val Phe Phe Pro Leu Ala Val Lys Asn Leu Leu Ile Asp Tyr val Leu Leu Gly Gly Leu Ile Ile val Phe Gin Cys Thr Leu Leu Ile Glu Ile Thr Ala Ile Tyr Tyr Gin Lys Lys Thr Pro Trp Gly Phe Arg Leu Pro Leu Ala Leu Val val val Ile val Val Met Ala Ile His Ile Phe Gly Thr Leu Ala Ser Tyr Trp Leu Phe Pro Val Leu Ile Ala Ile Ala Phe Leu Leu Pro Gin Lys Asp Asn Leu Leu Thr Ile Thr Ile Ile Ile Pro Ala Ser Ile Trp val Leu Ile Pro His Gin Thr Ala Glu Val Thr Leu Arg Phe Ser Leu Ala Ile Ser Ala Cys Ala Ala Ile Met Tyr Val Val Val Asp Ala Ile Arg Lys Leu His Thr Glu Leu Phe Tyr Leu Ser Thr Arg His Ala Leu Thr Gly Thr Leu Asn Arg His Gin Leu Asp Gly Phe Leu Lys Lys Cys Leu Arg His Arg Gin Leu Ala Asn Glu Ser Ala Val Ile Ala Val Ile Asp Ile Asp His Phe Lys Ser Val Asn Asp Leu Tyr Gly His Asp Thr Gly Asp Lys val Ile Thr Gin val val Glu Ile met Asn Thr His Cys Arg Glu Leu Asp Leu Leu Phe Arg Leu Gly Gly Asp Glu Phe Leu Leu Leu Phe Glu Asn Thr Ser Leu Thr Asp Ala Thr Leu Val Met Ser His Ile Gly Cys Arg Ile Gin Gin Thr His Tyr Pro Tyr His Ala Lys val Thr val Ser val Gly Leu Ala Glu Ala Leu Arg Thr Asp Asp Pro Glu Gin Trp Phe Lys Arg Ala Asp Gin Ala Leu Tyr His Ser Lys Lys met Gly Lys Asn Arg val Ser Phe Asp Glu Glu His val Ile Glu Leu Asn Gly Asp His Cys His Ala Leu Leu Gly Ser Thr His Gly His Arg <210> 67 <211> 1386 <212> DNA
<213> vibrio cholerae <400> 67 atggatcatc gcttttcgac caaactgttt ctgcttctca tgattgcttg gccgctttta 60 ttcggatcaa tgagtgaggc tgtagagcgc caaaccttga ctattgccaa ctcaaaagca 120 tggaaaccct attcttattt ggatgaacag ggacagcctt ctggcatatt gattgatttt 180 tggttggctt ttggtgaagc gaatcatgtc gatattgaat tccaactgat ggattggaat 240 gattccctag aagcggtgaa gcttggcaaa tccgatgttc aagctggttt gatccgttct 300 gcttcaagat tagcgtatct cgattttgca gaacctttac tgacaatcga tacacaactc 360 tacgtacacc gcacgttatt gggcgataaa ttggatacgc tgctatcggg ggccattaac 420 gtctcattag gtgtagtaaa agggggattt gaacaagagt tcatgcaacg agaatatcct 480 caacttaagt tgattgagta cgccaacaat gaattgatga tgtctgcagc aaagcgacga 540 gaattagatg gttttgtggc cgatactcag gtcgccaatt tctatatagt ggtttccaat 600 ggcgcgaaag attttacgcc agtgaagttt ctttattcag aggaattacg tccagcggtc 660 gccaaaggca atagggattt attagagcaa gtagagcagg ggtttgcaca attaagtagc 720 9ZE <TTZ>
69 <OTZ>

096 1)4111eDD6 63epD5D636 u3)61.1ueeu 1.66Dee6p61 616136uppp 61e1D61Dpp 006 6166pplpp6 601.661PD6P 6PPP3D6DP1 le1.611636e pule6)361p D3P6D1D11P
0V8 DDP133P1.11 Pa3P61666D 366eD13v31 D614111.661 D636u311.36 .613ep6166 08Z 34PP6P6333 llaeel.e336 Je6Dpau6eo eeD16D311) 666u DPPD6PPD1P
OZL PPDPDD6P61 PP166D6EPD alallaralx 6163116p6u p616616611.
099 peD1D11.31.6 D3p66D1.11P 13up6p361.p 6p36eD1D6D 1161E11D61 Dllpup61E6 009 16611p61p) p3p6631.1DE 31p6lepple 636puE11.11 pppeblleau 661161E636 OVS 31P661313P PP3PD1PP6P 6pe6EDu631 D331PED1P) D6111.D3PDP DD6D614P6D
08V pubpu5p1.16 DEp6D1631E pplprlle66 DppEolD6mul. p636D616 e6p61.p3614 OZV 11e61663e3 ullpul6pDp 111pppp636 111p663ppp 1p61161p36 plplappep6 09E 1)31u6Ppul eppappleul. 13616136p6 EADD11.11D pppuftp6u1 eEoplap6D3 00E 3611.11.361p p31.plue336 61up6D6pup uppDp111.16 7661PPP66D ITPD1D1DPD
OVZ 6u3DE1.1661. pepp6pD616 ael6pu3Dp6 pplupp6616 pppel6p66e preu166e36 08T 1361xeueup 3ellelluel 1163u36616 61P1.1.appep pal6op6611 Dleppeplla OZT Pl1DDP6D66 DEDDI.D4D6D DP11514PP6 1D1611DDP6 D661411P16 ppealEmplp 09 p366136D11 ueepu6eDET 61ppl1PPPE, 11e1166D46 e611)616pp Eop6lee6le 89 <00V>
DPJaL04D OJA <ETZ>
vNa <ZTZ>
186 <LIZ>
89 <OTZ>

661.D3e1.631 11)161166p papp6upp66 leu6636euP 66661 ep1136pleb D361u63E36 111.6upalep )66D136v3D Dupplpppla lzeD616136 ElulDpbeple DDUD1DPD1.6 1D1D1DPDPP D3D61PP616 P166616PPP PPPP6P3611 661 llpe6D6p61 DET1.1.6p36e 36D16P6PP6 P361PPPPP1 66163636P1 611P1A111 ou 6p6pp61661 6611p66pEo 61.113p1DDE 1.6631.ppEre 11136puprb D616161666 11.66163111 61.D1p3D61E plu666p36 D661e36661 11DEolpEop Pl1PDDPPPP
OZOT
Dllpe61P61. le1P611111 661e6116pp Pl1D6ED66P DI.P6DP3P2P DeE6636up6 11Dzepee61. 1.111163116 DApluulD6 lluDe66ou6 11.36p1x66D eppv6D6e11.

1P1DD1.31DP PUPPD1PPDD 6PP6PP611D PPD6PDPDPP 6D611P6D11 6DDPa6D663 elp6u31131 pelelllelz 631p16E.D1D el431.)466e plza6plall e6Dpoluell De21.6Deppa ellae6Depu 61221up11.5 664u6Da5ru lalapa6pDp epup6p61pu <212> PRT
<213> vibrio cholerae <400> 69 Met Asn Asp Lys Val Leu Glu Ser val Ile Glu Ile Thr Glu Gin Lys Asn Ser Leu Ala Leu Ser Tyr Ser Ile Leu Ala Thr Leu Ser Glu Leu Leu Pro Leu Ser Thr Ala Thr Leu Phe His His Leu Gly Arg Ser Thr Leu Met Val Ala Arg Leu Ile Ile Thr Lys Asn Ala Ala Gly Lys Lys Glu Tyr Gin Trp Gin Tyr Asp Gin Val Cys Ala Asp Asn Gly Tyr Gin His ser Gin Ser Glu met Ala Phe Ser Gin Gin Ala Asn Gly Gin Tyr Gin Cys Phe Cys Pro Ile Pro Ile Glu Glu His Phe Ser Ala Glu Leu Cys Leu Ile Leu Asn Lys Asp Pro Glu Pro Tyr Arg Met Leu Ile Asn Gly Phe Ala Lys Ile Tyr Arg Asn Tyr Thr Val Ile Leu His Glu Ser Glu Arg Asp Lys Leu Thr Gly Leu Leu Asn Arg Arg Thr Leu Glu Asp Arg Leu Arg His Thr Phe Ala Ile Asn Pro Ser Thr Glu Glu Asn His Lys Leu Trp Ile Ala Met Leu Asp Ile Asp His Phe Lys Ala Ile Asn Asp His Phe Gly His Met Ile Gly Asp Glu Ile Leu Leu Met Phe Ala Gin Gin Met Gin His Tyr Phe Gly Pro Ser Ser Gin Leu Phe Arg Phe Gly Gly Glu Glu Phe val Ile Ile Phe Ser Ser Gly Asn Glu Pro Gin Ile Lys Gin Gin Leu Asp Gly Phe Arg Gin Gin Ile Arg Arg His Asn Phe Pro Arg Ile Gly Glu Leu Ser Phe Ser Ala Gly Phe Cys Ser Leu Arg Pro Gly Asp Tyr Leu Pro Thr Ile Leu Asp His Ala Asp Lys Ala Leu Tyr Tyr Ala Lys Glu His Gly Arg Asn Gln val His Cys Tyr Glu Gin Leu Cys Glu Asn Gly Lys Ile Ala Ser Ala Gin Arg Pro Phe Ser Asp Asp Val Glu Leu Phe <210> 70 <211> 1311 <212> DNA
<213> Vibrio cholerae <400> 70 gtgagaatga cttggaactt tcaccagtac tacacaaacc gaaatgatgg cttgatgggc 60 aagctagttc ttacagacga ggagaagaac aatctaaagg cattgcgtaa gatcatccgc 120 ttaagaacac gagatgtatt tgaagaagct aagggtattg ccaaggctgt gaaaaaaagt 180 gctcttacgt ttgaaattat tcaggaaaag gtgtcaacga cccaaattaa gcacctttct 240 gacagcgaac aacgagaagt ggctaagctt atttacgaga tggatgatga tgctcgtgat 300 gagtttttgg gattgacacc tcgcttttgg actcagggaa gctttcagta tgacacgctg 360 aatcgcccgt ttcagcctgg tcaagaaatg gatattgatg atggaaccta tatgccaatg 420 cctatttttg agtcagagcc taagattggt cattctttac taattcttct tgttgacgcg 480 tcacttaagt cacttgtagc tgaaaatcat ggctggaaat ttgaagctaa gcagacttgt 540 gggaggatta agattgaggc agagaaaaca catattgatg taccaatgta tgcaatccct 600 aaagatgagt tccagaaaaa gcaaatagct ttagaagcaa atagatcatt tgttaaaggt 660 gccatttttg aatcatatgt tgcagattca attactgacg atagtgaaac ttatgaatta 720 gattcagaaa acgtaaacct tgctcttcgt gaaggtgatc ggaagtggat caatagcgac 780 cccaaaatag ttgaagattg gttcaacgat agttgtatac gtattggtaa acatcttcgt 840 aaggtttgtc gctttatgaa agcgtggaga gatgcgcagt gggatgttgg aggtccgtca 900 tcgattagtc ttatggctgc aacggtaaat attcttgata gcgttgctca tgatgctagt 960 gatctcggag aaacaatgaa gataattgct aagcatttac ctagtgagtt tgctagggga 1020 gtagagagcc ctgacagtac cgatgaaaag ccactcttcc caccctctta taagcatggc 1080 cctcgggaga tggacattat gagcaaacta gagcgtttgc cagagattct gtcatctgct 1140 gagtcagctg actctaagtc agaggccttg aaaaagatta atatggcgtt tgggaatcgt 1200 gttactaata gcgagcttat tgttttggca aaggctttac cggctttcgc tcaagaacct 1260 0081 allrlaa.Dr1 PETEITDPPP rfteur11Da. PelEDP1PPP 61D661D6D PDDPDD6261 OVLI 6166rDrlar 6661D616rr rDDD1DD661 aDDEIDDD6DD ZU61.614)DU laDDI.Dle6D
0891 1D1661p66u DD6e1161rD DeD1116666 1r6erulbra aaaar16111 llarr1D66D
OZ91 DIXDPDDPDD 5DDDE0166PD P1DE6661D6 PlE0P6DD11D D6rD1DD61D D1D11eDD61.
09S1 pD116661DD 11D61D1D6r rD6aDeD1D6 611D1r61rD 6616rDD16u 661.D66rDDD
00ST 6D161D1D6D 1D16r661eD eD111D11.DD 61Derre616 1rD1r11D11 DI.Daaeuele OVVT 661DarDere eurDrourel 1166eru6 61D6rDpe6 paall.DDrar re6are1Drr 08E1 erall6aar6 rur66rD611. 6arrr6e6re 6PPEDEEPP6 Z61D616DPE rr1D1rurr6 OZET 61rDlErDur 61111PPP66 PPPP6DZPDP DI.D1)11DDI. P1D66D661E DPPPETE6PP
09Z1 DD111661rr 266rr66rur D61PDEIPPDD DP1611D]Pl. 111PDAPPE 1Dr6Dr1Dur 6rr66r116 PPPPAPD11 1D661DPREP D11PAD61.3 Dffee6UPDD DED6P1D61.) OKI D661D6r16r PPPPD1PP66 111D661DDD PE1P1P6616 1D1P1PPPPE P616P11E11 0801 D11DuDre1.6 1D6aDDD6r6 66E66r6eur r66r6er6ar D1rD161r6r Dear6ruull OZOT PDPEoPP11P PE6PP66PPI. 1PD1PPEP66 P1126PEPD4 61D61P6Pel DlaD6rDlea 096 lulrer6166 er6e111.116 ED16P61D1D D1PPPP5PPP 6DDI.EPP6PE UP11.1EPP61 006 6a1aDu)4ea eD616D1DrD eeDD11.rleu 6ur6raDrED 11.PP6EDDDD l6PE661DEE
OVS P11161.PD16 le6111eP61. PelDDE061D 1.11e6re616 DrD6r61r1D eaD6r666DD
08L pDre61D6aD 666D.6)66 62D 6)6)) aDr6D616er 61Dr6rDaD6 1D61)DPDDP
OZL 661.6116666 rur61661.r6 666D66D66D rDD1D1r1r6 1e6D6DD6rD 1D6rr6116u 099 e6r6611.116 6D666DD1D6 re6D1DD666 6.D6666 ar666r66Du 16D1D1auDD
009 D66D1D16 6DD61.DD66D DDDETDDDEll 6DP6661DDD 666DD5DD1 DDU6UPDDET
OVS P6DPDDI.D61. 3636D6D666 6E6PD6DD6 1.1)11661A 66PDD111D1 366DAP666 08V D1.D66D61 1DD6r661D6 6666666 6aDDDD6D6r DyeDD6De61 D16DD6rD6D
OZV PDPMEDDA ADETD1.3DD DEIEIPPPPPDD 6D6DDA666 1DEPAADD 16DD6DD66 00E 16urr666D6 6eraDD61DD D6DD66r6DD DDD61D66DD 1D1.6r6DDeD DD1r661r6D
OVZ DDD6D6666r DD61ur66DE D6D1166r PDDDDA1DP DAP66D66 r6DDaaD6E6 r6ED61rDD6 6666 61aDD6eD61 eDD6eDD6D6 e66 663111DalD
OZT lar66Dree6 appallaDD6 6D6a1DaDD1 aD1D661D61 D6aD6rDrrD Dlaa661.1D6 09 1.6r1.6rDD6D DD6161.61.1.D aDr6rD6D16 66D11DDDD 166661DD6r DDD11Dr6D6 IL <00t>
suavies owoH <ETZ>
vNa <ZTZ>
TOZZ <FEZ>
TL <OTZ>
TTET r 6)6616era 66areDeD6r DETDITERET P6aDDETP5D 1.7D6PDal5P

taatatacgt gactaaatgt aaaacctaac ttattttctg ttatctattt atttttactt 1860 tcagtaacac tttttttatt ttaggtagca ttcagcctag aggcaactgc tgtttgttaa 1920 atatttcctg ttcatatatt ttgcacattt tcttatgggt tagttttctt ctcattgttt 1980 tgggaagttc ttaatatatt tggggtattt atctttcatt cgttgtctgt gtaacaaata 2040 acttctgcca tatgggttgt ctgcacattt tttggtgtct tttagtaaac aaggtttttt 2100 tgttttgtat tgttttgttt attgtaaaga tttttaaatt ttaatggagt tgatttcttt 2160 tctcattcaa gcttttgaga ataaattgga gttgaatttt t 2201 <210> 72 <211> 497 <212> PRT
<213> Homo sapiens <400> 72 Met Gln Pro Trp His Gly Lys Ala Met Gln Arg Ala Ser Glu Ala Gly Ala Thr Ala Pro Lys Ala Ser Ala Arg Asn Ala Arg Gly Ala Pro met Asp Pro Thr Glu Ser Pro Ala Ala Pro Glu Ala Ala Leu Pro Lys Ala Gly Lys Phe Gly Pro Ala Arg Lys Ser Gly Ser Arg Gln Lys Lys Ser Ala Pro Asp Thr Gln Glu Arg Pro Pro Val Arg Ala Thr Gly Ala Arg Ala Lys Lys Ala Pro Gln Arg Ala Gln Asp Thr Gln Pro Ser Asp Ala Thr Ser Ala Pro Gly Ala Glu Gly Leu Glu Pro Pro Ala Ala Arg Glu Pro Ala Leu Ser Arg Ala Gly Ser Cys Arg Gln Arg Gly Ala Arg Cys Ser Thr Lys Pro Arg Pro Pro Pro Gly Pro Trp Asp Val Pro Ser Pro Gly Leu Pro Val Ser Ala Pro Ile Leu Val Arg Arg Asp Ala Ala Pro Gly Ala Ser Lys Leu Arg Ala Val Leu Glu Lys Leu Lys Leu Ser Arg Asp Asp Ile Ser Thr Ala Ala Gly Met Val Lys Gly Val Val Asp His Leu Leu Leu Arg Leu Lys Cys Asp Ser Ala Phe Arg Gly Val Gly Leu Leu Asn Thr Gly Ser Tyr Tyr Glu His Val Lys Ile Ser Ala Pro Asn Glu Phe Asp Val met Phe Lys Leu Glu Val Pro Arg Ile Gin Leu Glu Glu Tyr Ser Asn Thr Arg Ala Tyr Tyr Phe Val Lys Phe Lys Arg Asn Pro Lys Glu Asn Pro Leu Ser Gin Phe Leu Glu Gly Glu Ile Leu Ser Ala Ser Lys met Leu Ser Lys Phe Arg Lys Ile Ile Lys Glu Glu Ile Asn Asp Ile Lys Asp Thr Asp Val Ile Met Lys Arg Lys Arg Gly Gly Ser Pro Ala Val Thr Leu Leu Ile Ser Glu Lys Ile Ser Val Asp Ile Thr Leu Ala Leu Glu Ser Lys Ser Ser Trp Pro Ala Ser Thr Gin Glu Gly Leu Arg Ile Gin Asn Trp Leu Ser Ala Lys Val Arg Lys Gin Leu Arg Leu Lys Pro Phe Tyr Leu Val Pro Lys His Ala Lys Glu Gly Asn Gly Phe Gin Glu Giu Thr Trp Arg Leu Ser Phe Ser His Ile Glu Lys Glu Ile Leu Asn Asn His Gly Lys Ser Lys Thr Cys Cys Glu Asn Lys Glu Glu Lys Cys Cys Arg Lys Asp Cys Leu Lys Leu Met Lys Tyr Leu Leu Glu Gin Leu Lys Glu Arg Phe Lys Asp Lys Lys His Leu Asp Lys Phe Ser Ser Tyr His Val Lys Thr Ala Phe Phe His Met Glu Ser Arg Ser val Ala Gin Ala Gly Val Gin Trp His AS Leu Gly Ser Leu Gin Ala Leu Leu Pro Gly Phe Met Pro Phe Ser Cys Leu Ser Leu Pro Ser Ser Trp Asp Tyr Arg Cys Pro Pro Pro His Pro Ala Asn Phe Leu Tyr Phe <210> 73 <211> 1071 <212> DNA
<213> Peptoclostridium difficile <400> 73 atggagaatt ttctagataa taaaaatatg ctatatgcat taaaaatgat atctcctgga 60 actccactta gattaggtct aaacaatgta ctaagagcta agactggtgg attaattgta 120 attgcaacaa acgaagatgt aatgaaaata gtagatggag gatttgctat aaatgcagaa 180 tattcaccat catatctata tgaattagct aaaatggatg gagctatagt tttaagtggt 240 gatgtaaaga aaatattatt tgctaatgca caacttatac ctgactattt tatagaaaca 300 tcagagacag gaacaagaca tagaacagca gaaagagtag caaaacaaac tggtgctata 360 gtcataggaa tttcacaaag aagaaatgtt ataacagttt atagaggaaa tgagaagtat 420 gtagtcgaag atatatctaa gatatttact aaggcaaatc aggctataca aactctggaa 480 aaatataaga cagtattgga ccaagctgta acaaatttaa atgccttaga gtttaatgat 540 ttggtaacta tttatgatgt tgcattagtc atgcaaaaga tggaaatggt aatgagagtt 600 acaagtataa ttgaaaaata tgtgatagaa ttgggtgatg aaggaacttt agtaagtatg 660 caattagaag aattaatggg tacaaccaga atagaccaga aattaatatt caaagattat 720 aataaagaaa acacagaaat aaaagaactt atgaaaaagg tcaaaaattt aaattcagaa 780 gaactaatag aattggttaa tatggcaaaa ctattagggt atagtggttt ttCagaaagt 840 atggatatgc ctataaaaac aagaggttat aggattctta gcaaaataca tagactacca 900 acagcaataa tagaaaactt agtaaattat tttgaaaact ttcaacaaat tttagatgca 960 tctattgaag aattagatga ggttgaagga ataggtgaaa taagagcaac atatataaaa 1020 aatggactca taaaaatgaa acaattagtc ttattagata gacacatatg a 1071 <210> 74 <211> 1083 <212> DNA
<213> Bacillus subtilis <400> 74 atggaaaaag agaaaaaagg ggcgaaacac gagttagacc tgtcatctat attgcagttt 60 gttgctccgg gtacaccgct cagagcgggg atggaaaacg tcttgagagc aaatacaggc 120 ggtctgattg ttgttggata taatgataaa gtaaaagaag tggtggacgg cggctttcac 180 ataaacacgg ctttttctcc ggcgcattta tatgagctgg ctaaaatgga tggagcgatc 240 attttaagtg attctggtca aaagatccta tacgcgaata ctcagctgat gccggatgcc 300 acaatttctt catcagaaac aggaatgcgg cacagaactg ccgaaagagt agctaagcaa 360 actggctgtc ttgtaatcgc catttctgaa agaagaaatg tcataacgtt atatcaggaa 420 aacatgaagt atacactaaa agacatagga tttattttaa ccaaggcgaa ccaagccatt 480 caaacacttg aaaaatataa gacaatcctc gataaaacga ttaatgcact gaacgcgtta 540 gagtttgagg aacttgttac cttcagtgat gtcttgtctg tcatgcatcg ttatgaaatg 600 gtactgagaa tcaaaaacga aattaatatg tatatcaaag agctggggac agaagggcat 660 ctgatcaaac tgcaagtcat tgaattgatt acggatatgg aagaagaggc cgctttattt 720 attaaggact atgtaaaaga aaagattaaa gatccgtttg ttctcttgaa ggagctgcag 780 gatatgtcca gttatgatct gctggatgat tccattgtgt ataagcttct cggttaccct 840 gcttctacta atcttgatga ttatgtattg ccgagaggat acaggctgtt aaataagata 900 ccgcgtcttc cgatgccgat tgttgaaaat gttgtagaag catttggagt cctgccaagg 960 attattgagg cgagtgcaga agaattagat gaagtagagg gaatcggtga agtacgagcc 1020 caaaaaatca aaaaaggatt aaaacgcctg caagagaagc attatttaga cagacaactg 1080 tga 1083 <210> 75 <211> 360 <212> PRT
<213> Bacillus subtilis <400> 75 Met Glu Lys Glu Lys Lys Gly Ala Lys His Glu Leu Asp Leu Ser Ser Ile Leu Gin Phe Val Ala Pro Gly Thr Pro Leu Arg Ala Gly Met Glu Asn Val Leu Arg Ala Asn Thr Gly Gly Leu Ile Val Val Gly Tyr Asn Asp Lys val Lys Glu val val Asp Gly Gly Phe His Ile Asn Thr Ala Phe Ser Pro Ala His Leu Tyr Glu Leu Ala Lys Met Asp Gly Ala Ile Ile Leu Ser Asp Ser Gly Gln Lys Ile Leu Tyr Ala Asn Thr Gin Leu Met Pro Asp Ala Thr Ile Ser Ser Ser Glu Thr Gly Met Arg His Arg , Thr Ala Glu Arg Val Ala Lys Gin Thr Gly Cys Leu Val Ile Ala Ile Ser Glu Arg Arg Asn Val Ile Thr Leu Tyr Gin Glu Asn met Lys Tyr Thr Leu Lys Asp Ile Gly Phe Ile Leu Thr Lys Ala Asn Gin Ala Ile Gin Thr Leu Glu Lys Tyr Lys Thr Ile Leu Asp Lys Thr Ile Asn Ala Leu Asn Ala Leu Glu Phe Glu Glu Leu Val Thr Phe Ser Asp Val Leu Ser val Met His Arg Tyr Glu Met Val Leu Arg Ile Lys Asn Glu Ile Asn Met Tyr Ile Lys Glu Leu Gly Thr Glu Gly His Leu Ile Lys Leu Gin Val Ile Glu Leu Ile Thr Asp Met Glu Glu Glu Ala Ala Leu Phe Ile Lys Asp Tyr Val Lys Glu Lys Ile Lys Asp Pro Phe Val Leu Leu Lys Glu Leu Gin Asp Met Ser Ser Tyr Asp Leu Leu Asp Asp Ser Ile Val Tyr Lys Leu Leu Gly Tyr Pro Ala Ser Thr Asn Leu Asp Asp Tyr Val Leu Pro Arg Gly Tyr Arg Leu Leu Asn Lys Ile Pro Arg Leu Pro met Pro Ile val Glu Asn val Val Glu Ala Phe Gly val Leu Pro Arg Ile Ile Glu Ala Ser Ala Glu Glu Leu Asp Glu Val Glu Gly Ile Gly Glu val Arg Ala Gin Lys Ile Lys Lys Gly Leu Lys Arg Leu Gin Glu Lys His Tyr Leu Asp Arg Gin Leu <210> 76 <211> 454 <212> PRT
<213> Pelobacter propionicus , <400> 76 Met Arg Arg Ile Leu Val Val Glu Asp Asp Arg Phe Phe Arg Asp Leu Phe Tyr Asp Leu Leu Val Gly Gin Gly Tyr Asp Val Asp Arg Ala Ser Ser Gly Glu Glu Gly Leu Asp Arg Leu Ser Thr Tyr Ala Phe Asp Leu Val Val Thr Asp Leu Val Met Pro Gly val Asp Gly Met Asp Ile Leu Ala Arg Ala Arg Glu Asn Asp Pro Ser Ala Asp val Ile Met Val Thr Gly Asn Ala Asn Leu Glu Ser Ala Ile Phe Ala Leu Lys His Gly Ala Arg Asp Tyr Phe Val Lys Pro Ile Asn Pro Asp Glu Phe Leu His Ser val Ala Gin Cys Leu Glu Gin Arg Arg Ile Leu Asp Glu Asn Glu Glu Leu Lys Ser Met Leu Asn Leu Tyr Gin Ile Ser Gin Ala Ile Ala Gly Cys Leu Asp Met Glu Arg Leu Gin His Leu Ile Phe Asp Ala Phe Thr Arg Glu Ile Gly Thr Ser Arg Gly Met Cys Leu Phe Ala Thr Glu Thr Gly Leu Glu Leu Cys Glu Val Lys Gly val Glu Thr Ala Val Ala Glu Arg Cys Ile Ala Ser Val Leu Glu Arg Leu Ser Glu Asp His Pro Asp Glu Cys Asn Ser Leu Arg Ile Ser Phe Gin Gly Gly Gly Asp Asp Ser Gly Ile Glu Ala Ala Ile Leu Ile Pro Leu Arg Gly Lys Gly Ser Gin Arg Gly Val Val Val Ala Phe Asn Glu Pro Gly Leu Gly Leu Pro Glu Leu Gly Ala Arg Lys Lys Asn Ile Leu Phe Leu Leu Glu Gin Ser Leu Leu Ala Leu Glu Asn Ala Ser Ser Tyr Ser Leu Ala Lys Asp Met Leu Phe Ile Asp Asp Leu Ser Gly Leu Tyr Asn Gln Arg Tyr Leu Glu Val Ala Leu Glu Arg Glu Met Lys Arg Ile Gly Arg Phe Ser Ser Gin Leu Ala Val Leu Phe Leu Asp Met Asp Ser Phe Lys Gin Val Asn Asp Thr His Gly His Leu Val Gly Ser Arg Val Leu Lys Glu Met Gly Thr Leu Leu Arg Leu Ser Val Arg Asp Val Asp Val Val Ile Arg Tyr Gly Gly Asp Glu Tyr Thr Ala Ile Leu Val Glu Thr Ser Pro Ala Ile Ala Ala Asn Val Ala Glu Arg Ile Arg Ser Met Val Ala Ser His Val Phe Leu Ala Asp Glu Gly Tyr ASp Ile Arg Leu Thr Cys Ser Ile Gly Tyr Ser Cys Cys Pro Glu Asp Ala Leu Thr Lys Glu Glu Leu Leu Glu Met Ala Asp Gin Ala Met Tyr Thr Gly Lys Gly Arg Gly Lys Asn Cys val val Arg Phe Thr Lys Thr Ser <210> 77 <211> 462 <212> PRT
<213> Geobacter uraniireducens <400> 77 Met Glu Arg Ile Leu val val Glu Asp Asp Ser Phe Phe Arg Glu val Phe Ala Asp Leu Leu Ile Glu Asp Gly Phe His Val Asp Val Ala Ala Ser Gly Glu Gin Ala Leu Val Met Val Gin Asn Arg Glu Tyr Gin Leu val val Thr Asp Leu Val Met Pro Asp Ile Thr Gly Leu Asp Ile Leu Ser Lys Val Lys Gin Leu Asp Pro Thr Ile Asp Val Ile met val Thr Gly His Ala Asn Met Glu Thr Ala Ile Phe Ala Leu Lys Asn Gly Ala Arg Asp Tyr Leu Val Lys Pro Ile Asn His Asp Glu Phe Lys His Ala Val Ala Leu Cys Phe Glu Gin Arg Arg Leu Leu Asp Glu Asn Gin Glu Leu Lys Gly Leu Ile Asn Leu Tyr His Val Ser Gin Thr Ile Ala Asn Cys Leu Asp Leu Glu Arg Ile His Thr Leu Leu Val Asp Ser Leu Ala Lys Glu Phe Ala Val Ser Arg Gly Leu Gly Tyr Phe Leu Asp Gly Ala Asp Asn Leu_Glu Leu Lys Ala Leu Lys Gly Val Ser Glu Ala Ser Ala Gly Arg Leu Gly Glu Leu Ile Leu Ser Arg Tyr Asn Val Gin Gly Glu Asp Ser Arg Ser Phe Val Leu Leu His Asp Phe Met Gin Pro Asp Ala Asp Phe Gly Leu Gly Thr Asp Gly Asp Met Lys Glu Ala Met Leu Phe Phe Val Arg Ser Arg Thr Val Leu Gin Gly Ile Val Ile Leu Phe Ser Glu Pro Gly Thr Ser Phe Pro Ala Asp Ile Gin Phe Lys Asn Ile Asn Phe Leu Leu Asp Gin Ser Ser Leu Ala Leu Glu Asn Ala Val Arg Tyr Asn Asn Ala Lys Asn Leu Leu Tyr Ile Asp Glu Leu Thr Gly Leu Phe Asn Tyr Arg Tyr Leu Asp Val Ala Leu Glu Arg Glu Ile Arg Arg Ala , Glu Arg Tyr Gly Ser His Ile Ser Val Ile Phe Leu Asp Ile Asp Leu Phe Lys Arg Val Asn Asp Met Tyr Gly His Leu val Gly Ser Arg Ala Leu Asn Glu Val Gly Ile Leu Leu Lys Lys Ser Val Arg Asp val Asp Thr Val Ile Arg Tyr Gly Gly Asp Glu Tyr Thr Ile Ile Leu Ile Glu Thr Gly Ile Asp Gly Ala Ala Ala val Ala Glu Arg Ile Arg Arg Ser Ile Glu Ala His Gly Phe Met Ala Ala Asp Gly Leu Asn Leu Lys Leu Thr Ala Ser Leu Gly Tyr Ala Cys Tyr Pro Glu Asp Ala Lys Thr Lys Thr Glu Leu Leu Glu Leu Ala Asp Gin Ala Met Tyr Arg Gly Lys Ala Asp Gly Lys Asn Arg Val Phe Tyr Val Ser Ala Lys Asn Asn <210> 78 <211> 460 <212> PRT
<213> Geobacter daltonii <400> 78 Met Glu Arg Ile Leu Val Val Glu Asp Asp Ser Phe Phe Arg Glu Val Phe Ala Asp Leu Leu Arg Asp Asp Gly Phe Ala val Asp Val Ala Cys Ser Gly Glu Lys Ala Leu Glu met Leu Arg Ser Ser Glu Tyr Ala Leu Val Val Thr Asp Leu Val Met Pro Asp Ile Thr Gly Leu Asp Leu Leu Ser Lys Val Lys Gin Phe Asp Pro Ser Ile Asp Val Ile Leu val Thr Gly His Ala Asn Thr Glu Thr Ala Val Phe Ala Leu Lys Asn Gly Ala Arg Asp Tyr Leu Val Lys Pro Ile Asn Ser Glu Glu Phe Lys His Ala , , val Ala Leu Cys Phe Glu Gin Arg Arg Leu Leu Asp Glu Asn Gin Glu Leu Lys Gly Leu Leu Asn Leu Phe Gin Ile ser Gin Thr Ile Ala Asn Ser Leu Asp Phe Asp Arg Ile His Thr Ile Leu val Asp Ser Leu Ala Lys Glu Phe Gly Leu Ser Arg Leu Thr Gly Tyr Phe Gin Asn Asp Asp Gly Thr Leu Glu Leu Lys Glu Ile Lys Gly Phe Asp Glu Glu Thr Ala Ser Ser Leu Gly Glu Leu Ile Phe Asp Ile Phe Asp Val Arg Glu Glu Asp Asn Arg Ser Phe Val Leu Leu Asn Asp Leu Glu Gin Arg Ser Arg Phe Phe Ala Glu His Ser Val Thr Glu Ala met Leu Phe Phe Val Arg Ala Lys Thr Ala Leu Leu Gly Ile Ile Ile Val Phe Asn Glu Ser Gin Ser Val Phe Pro Ala His Leu Asp Phe Lys Asn Ile Asn Phe Leu Leu Asp Gin Ala Ser Leu Ala Leu Glu Asn Ala Ser Arg Tyr Asn Asn Ala Lys Asn Leu Leu Tyr Tie Asp Glu Leu Thr Gly Leu Phe Asn Tyr Arg Tyr Leu Asp Val Ala Leu Glu Arg Glu val Arg Arg Ala Glu Arg Tyr Ser Ser Asn Ile Ser Ile Ile Phe Leu Asp Ile Asp Leu Phe Lys Arg Ile Asn Asp Gin Tyr Gly His Leu Val Gly Ser Lys Ala Leu Ala Glu Val Gly Leu Leu Leu Lys Lys Ser val Arg Asp val Asp Thr Val Ile Arg Tyr Gly Gly Asp Glu Tyr Thr Ile Ile Leu Ile Glu Thr Gly Ile Asp Gly Ala Ser val val Ala Glu Arg Ile Arg Ser Thr Ile Glu Gly His Val Phe Ile Gin Ser Glu Gly Leu Asp Ile Lys Leu Thr Ala Ser Leu Gly Cys Ala Ser Tyr Pro Glu Asp Ala Cys Thr Lys Leu Glu Leu Leu Glu Leu Ala Asp Gin Ala met Tyr Arg Ser Lys Ala Cys Gly Lys Asn Met val Phe His Ile Ser Ala Tyr Lys Lys Gin <210> 79 <211> 4 <212> PRT
<213> unknown <220>
<221> source <223> /note="Description of unknown:
'DEAD' box family motif peptide"
<400> 79 Asp Glu Ala AS

<210> 80 <211> 8 <212> PRT
<213> Artificial Sequence <220>
<221> source <223> /note="Description of Artificial Sequence: Synthetic peptide"
<400> 80 Ser Ile Ile Asn Phe Glu Lys Leu <210> 81 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<221> source <223> /note="Description of Artificial Sequence: Synthetic primer"
<400> 81 ataggtaccc caccgtgatg acaactgaag atttca 36 <210> 82 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<221> source <223> /note="Description of Artificial Sequence: synthetic primer"
<400> 82 atactcgagt tagagcggca tgactcgat 29 <210> 83 <211> 40 <212> DNA
<213> Artificial Sequence <220>
<221> source <223> /note="Description of Artificial Sequence: Synthetic primer"
<400> 83 tgacagctta tcgttatgcc gctgaagagt ttgcactgat 40 <210> 84 <211> 5 <212> PRT
<213> Vibrio cholerae <400> 84 Gly Gly Glu Glu Phe <210> 85 <211> 5 <212> PRT
<213> Artificial Sequence <220>
<221> source <223> /note="Description of Artificial Sequence: Synthetic peptide"
<400> 85 Ala Ala Glu Glu Phe <210> 86 <211> 15 <212> PRT
<213> Artificial Sequence <220>
<221> source <223> /note="Description of Artificial Sequence: Synthetic peptide"
<400> 86 val Asn Gly Ser Arg Tyr Tyr Phe Asp Thr Asp Thr Ala Ile Ala <210> 87 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<221> source <223> /note="Description of Artificial sequence: Synthetic primer"
<400> 87 ataggtaccc caccatgaat gacaaagtgc t 31 <210> 88 <211> 34 <212> DNA
<213> Artificial Sequence <220>
<221> source <223> /note="Description of Artificial Sequence: Synthetic primer"
<400> 88 atactcgagt tagaaaagtt caacgtcatc agaa 34 <210> 89 <211> 50 <212> DNA
<213> Artificial Sequence <220>
<221> source <223> /note="Description of Artificial sequence: Synthetic primer"
<400> 89 gtcttctcaa ctatttcgct ttgctgctga agagttcgtg attatttttt 50 <210> 90 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<221> source <223> /note="Description of Artificial Sequence: Synthetic primer"
<400> 90 gctactacga ggacggcctg 20 <210> 91 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<221> source <223> /note="Description of Artificial Sequence: Synthetic primer"
<400> 91 ctcatcgatg atcagcttgc c 21 <210> 92 <211> 9 <212> PRT
<213> Artificial Sequence <220>
<221> source <223> /note="Description of Artificial Sequence: Synthetic peptide"
<400> 92 Ala Met Gin Met Leu Lys Glu Thr Ile <210> 93 <211> 26 <212> PRT
<213> Pseudomonas aeruginosa <400> 93 Leu Thr Asp Pro Leu Thr Gly Leu Pro Asn Arg Ala Ala Leu Ser Glu Arg Leu Glu Gin Glu Val Ala Arg Arg His <210> 94 <211> 14 <212> PRT
<213> Pseudomonas aeruginosa <400> 94 Leu Leu Ala Val Leu Asp Ile Asp His Phe Lys Arg Ile Asn <210> 95 <211> 25 <212> PRT
<213> Pseudomonas aeruginosa <400> 95 Asp Phe Gly His Leu Ala Gly Asp Lys Val Leu Lys Ile Ile Ala Gly Glu Leu Arg Lys Arg Leu Arg Gin Ala <210> 96 <211> 17 <212> PRT
<213> Pseudomonas aeruginosa <400> 96 Ile Ala Arg Phe Gly Gly Glu Glu Phe val Val Leu Leu Pro Ala Thr Ser <210> 97 , <211> 19 <212> PRT
<213> Pseudomonas aeruginosa <400> 97 Glu Ala Gly Arg Gin Leu Leu Glu Arg Leu Arg Ala Ala Ile Ala Ala Cys Pro Phe <210> 98 <211> 19 <212> PRT
<213> Pseudomonas aeruginosa <400> 98 Pro Leu Ser Ile Thr Cys Ser Ala Gly Ile Thr Ala Phe Glu Gly Asn Glu Ala Gly <210> 99 <211> 4 <212> PRT
<213> Pseudomonas aeruginosa <400> 99 Ala Val Phe Glu <210> 100 <211> 19 <212> PRT
<213> Pseudomonas aeruginosa <400> 100 Ala Asp Gin Ala Leu Tyr Arg Ala Lys Arg Ala Gly Arg Asp Arg Leu Glu Val Ala <210> 101 <211> 26 <212> PRT
<213> Pseudomonas aeruginosa <400> 101 Tyr Ile Asp Glu Pro Thr Gly Leu Tyr Asn Arg Leu Arg Leu Gin Glu Asp Val Ser Leu Arg Leu Gin Arg Asp Gly <210> 102 <211> 14 <212> PRT

, ' <213> Pseudomonas aeruginosa <400> 102 Thr val Ile Ala Ala Asp Leu Leu Pro Leu Ala Leu Leu Asn <210> 103 <211> 25 <212> PRT
<213> Pseudomonas aeruginosa <400> 103 Thr Leu Gly Tyr Pro Phe Ser Asn Asp Leu Met Leu Glu Ala Arg Asp Arg Ile Arg Ala Glu Leu Pro Asp Phe <210> 104 <211> 17 <212> PRT
<213> Pseudomonas aeruginosa <400> 104 Leu Tyr Lys Ile Ser Pro Thr Arg Phe Gly Leu Leu Leu Pro Arg Gin Gin <210> 105 <211> 19 <212> PRT
<213> Pseudomonas aeruginosa <400> 105 Glu Glu Thr Glu Ser val Cys Leu Arg Leu Leu Arg Ala Phe Glu Ser Pro val val <210> 106 <211> 15 <212> PRT
<213> Pseudomonas aeruginosa <400> 106 Lys Ala Asn Val Gly Leu Gly Val Leu Pro Leu Ala AS Asp Thr <210> 107 <211> 4 <212> PRT
<213> Pseudomonas aeruginosa <400> 107 Asp Trp Leu Arg <210> 108 <211> 19 <212> PRT
<213> Pseudomonas aeruginosa <400> 108 Val Val Ser Ala Ala Asp Asp Ala Arg Asp Arg Gly Val Gly Trp Ala Arg Tyr Asn <210> 109 <211> 26 <212> PRT
<213> Pseudomonas aeruginosa <400> 109 Ser Gin Asp Pro Val Thr Gly Leu Tyr Asn Arg Ser His Phe Leu Asp Leu Met Asp Ala Ala Val Gin Gin Ala val <210> 110 <211> 14 <212> PRT
<213> Pseudomonas aeruginosa <400> 110 Thr Leu Ala Tyr Ile His Leu Asn Gly Tyr Pro Ser Leu Gin <210> 111 <211> 25 <212> PRT
<213> Pseudomonas aeruginosa <400> 111 Asp His Gly Leu Ser Gly Ile Asp Leu Leu Leu Gly Gin Leu Ala Gly Leu Met Arg Glu Gin Phe Gly Glu Glu <210> 112 <211> 17 <212> PRT
<213> Pseudomonas aeruginosa <400> 112 Leu Ala Arg Phe Gly Asp Ser Ile Phe Ala Ala Leu Phe Lys Gly Lys Thr <210> 113 <211> 19 <212> PRT
<213> Pseudomonas aeruginosa <400> 113 Glu Gin Ala Gin Ala Ala Leu Gin Arg Leu Leu Lys Lys Val Glu Asn His Leu Phe <210> 114 <211> 15 <212> PRT
<213> Pseudomonas aeruginosa <400> 114 Gin Ala Thr Leu Ser Ile Gly Val Ala Gly Leu Asp Glu Lys Thr <210> 115 <211> 4 <212> PRT
<213> Pseudomonas aeruginosa <400> 115 Asp Val met Asn <210> 116 <211> 19 <212> PRT
<213> Pseudomonas aeruginosa <400> 116 Ala His Arg cys Ala Asp Asp Ala Ala Arg Lys Gly Gly Ser Gin Ile Lys Gin Tyr <210> 117 <211> 26 <212> PRT
<213> Salmonella enterica <400> 117 Met Arg Phe Pro val Ser Glu Leu Pro Asn Lys Ala Phe Leu Met Ala Leu Leu Glu Gin Val Ile Thr Arg Gin Gin <210> 118 <211> 12 <212> PRT
<213> Salmonella enterica <400> 118 Ala Leu Ile Ile Val Thr Cys Glu Thr Leu Arg Asp <210> 119 <211> 27 <212> PRT
<213> Salmonella enterica <400> 119 Ala Gly Val Leu Gin Glu Thr Gin Arg Glu Ile Leu Leu Leu Thr Leu val Glu Lys Leu Lys Ser Val Leu Ala Pro Arg <210> 120 <211> 17 <212> PRT
<213> Salmonella enterica <400> 120 Leu Thr Gin Val Ser Gly Tyr Asp Phe Ala Ile Ile Ala His Gly Val Lys <210> 121 <211> 19 <212> PRT
<213> Salmonella enterica <400> 121 Trp His Ala Ile Thr Leu Gly Gin Gin Ile Leu Thr Ile Ile Asn Glu Arg Leu Pro <210> 122 <211> 17 <212> PRT
<213> Salmonella enterica <400> 122 Gin Leu Arg Pro Ser Cys Ser Ile Gly Ile Ala Met Tyr Tyr Gly Asp Leu <210> 123 <211> 23 <212> PRT
<213> Salmonella enterica <400> 123 Leu Tyr Gly Arg Ala Val Ser Ala Ala Phe Thr Ala Arg Arg Lys Gly Lys Asn Gin Ile Gin Phe Phe <210> 124 <211> 26 <212> PRT
<213> Vibrio cholerae <400> 124 Ser His Asp Leu Gly Thr Gly Phe Leu Asn Arg Thr Ala Leu Glu Gin Gin Leu Ala Met Gin Leu Ala Gin Leu Ala <210> 125 <211> 14 <212> PRT
<213> Vibrio cholerae <400> 125 Ala Val Ile His Ile Gly Phe Ala Asn Ala Arg Gin Leu Gin <210> 126 <211> 25 <212> PRT
<213> Vibrio cholerae <400> 126 Arg Leu Gly Tyr His Leu Trp Asp Asp Val Leu Lys Gin Leu Arg Glu Arg Leu Gly Pro val Thr Glu Gly Glu <210> 127 <211> 17 <212> PRT
<213> vibrio cholerae <400> 127 Thr Ala Arg Pro Asn Ser Thr Asn Leu Thr Leu Ile Leu Lys Ala His Pro <210> 128 <211> 10 <212> PRT
<213> Vibrio cholerae <400> 128 Thr Gin Leu Asn Gin Leu Cys His Arg Leu <210> 129 <211> 9 <212> PRT
<213> Vibrio cholerae <400> 129 Ala Gly Gin Ala Gin Phe Val Thr Glu <210> 130 <211> 15 <212> PRT
<213> Vibrio cholerae <400> 130 His Leu Asn Pro Tyr Ile Gly Val Ala Leu Ser Arg Glu Thr Arg <210> 131 <211> 6 <212> PRT
<213> vibrio cholerae <400> 131 Pro Gln Gin Leu Leu Arg <210> 132 <211> 19 <212> PRT
<213> Vibrio cholerae <400> 132 Ala val Ser Ser Met Leu Ala Cys Lys Asp Ser Gly Tyr Lys Val Phe Phe His Ser <210> 133 <211> 26 <212> PRT
<213> xylella fastidiosa <400> 133 Gin Lys Ala Ser Val Thr Glu Leu Gin Thr Arg Thr His Ile Val Glu Gin Leu Glu Val Ala Leu Ser Ser Gly Ala <210> 134 <211> 14 <212> PRT
<213> Xylella fastidiosa <400> 134 Ala Leu Phe Phe Ile Glu val Ser Ser Ala Leu Asn Leu Arg <210> 135 <211> 25 <212> PRT
<213> xylella fastidiosa <400> 135 Arg Tyr Gly Tyr Thr Ala Phe Glu Arg Leu Met Asn Gin Val Glu Tyr His Leu Ala Gin Glu Ala His Pro Tyr <210> 136 <211> 17 <212> PRT
<213> xylella fastidiosa <400> 136 Leu Ala Arg Ile Ser Asp His Ser Phe Leu Leu Leu Ala Ile Asp Leu Ala <210> 137 <211> 10 <212> PRT
<213> xylella fastidiosa <400> 137 Glu His Gin Ala Leu Ala Ser His Leu Arg <210> 138 <211> 9 <212> PRT
<213> xylella fastidiosa <400> 138 Leu Ala Thr Leu Pro Leu Pro Ile Gin <210> 139 <211> 15 <212> PRT
<213> Xylella fastidiosa <400> 139 His Leu Arg Ser Ala Ile Gly Tyr Ala Pro Leu Asn Gin Gly Phe <210> 140 <211> 4 <212> PRT
<213> Xylella fastidiosa <400> 140 Asp Ala val Glu <210> 141 <211> 19 <212> PRT
<213> Xylella fastidiosa <400> 141 Thr Glu Arg Ala Thr Leu Glu Ala Arg Gin Asn Asn Glu Gly Ile Tyr Ala Tyr Val <210> 142 <211> 26 <212> PRT
<213> Xylella fastidiosa <400> 142 Gln Arg Asp Pro Ile Thr Gly Leu Leu Asn Arg Pro Ala Phe Ile Ile Ala Leu Glu Asp Ala val Ala Arg Ala Gly <210> 143 <211> 12 <212> PRT
<213> Xylella fastidiosa <400> 143 Gly Leu Leu Leu Val Glu Pro Asn His Tyr Thr Arg <210> 144 <211> 27 <212> PRT
<213> Xylella fastidiosa <400> 144 Leu Gin Glu Ile Gly Leu Asp Ser Ala Asp Thr Leu Ile Thr Ser Leu Ala Asn Phe Phe Ser Glu Met Ile Asp Ser Lys <210> 145 <211> 27 <212> PRT
<213> Xylella fastidiosa <400> 145 Thr Ala Arg Phe Cys Glu Thr Arg Phe Ala Leu Leu Leu Glu Gly Asp Tyr Thr His Thr Met Thr Leu Ala Glu Arg Ile <210> 146 <211> 9 <212> PRT

<213> Xylella fastidiosa <400> 146 Ile Asp Ile Ala Gin His Ile Phe Leu <210> 147 <211> 15 <212> PRT
<213> Xylella fastidiosa <400> 147 Thr Val Thr Val Ser Ile Gly Gly Val Gin Ile Gly Glu Arg Ile <210> 148 <211> 4 <212> PRT
<213> xylella fastidiosa <400> 148 Gin Val Leu His <210> 149 <211> 19 <212> PRT
<213> Xylella fastidiosa <400> 149 Ala Ala Glu Ser Val Gin Ile Ala Thr Gin Leu Giy Giy Asn Thr Ala Ile Ile Tyr <210> 150 <211> 26 <212> PRT
<213> Escherichia coli <400> 150 Met Arg Phe Pro Val Ser Asp Leu Pro Asn Lys Ala Leu Leu Met Glu Met Leu Glu Gin val val Ala Arg Lys Gin <210> 151 <211> 12 <212> PRT
<213> Escherichia coil <400> 151 Ala Leu Met he Ile Thr Cys Glu Thr Leu Arg Asp <210> 152 <211> 27 <212> PRT

, -<213> Escherichia coil <400> 152 Ala Gly Val Leu Lys Glu Ala Gin Arg Glu Ile Leu Leu Leu Thr Leu Val Glu Lys Leu Lys Ser Val Leu Ser Pro Arg <210> 153 <211> 17 <212> PRT
<213> Escherichia coli <400> 153 Leu Ala Gin Ile Ser Gly Tyr Asp Phe Ala Val Ile Ala Asn Gly Val Gin <210> 154 <211> 19 <212> PRT
<213> Escherichia coil <400> 154 Trp His Ala Ile Thr Leu Gly Gin Gin Val Leu Thr Ile Met Ser Glu Arg Leu Pro <210> 155 <211> 17 <212> PRT
<213> Escherichia coil <400> 155 Gin Leu Arg Pro His Cys Ser Ile Gly Val Ala Met Phe Tyr Gly Asp Leu <210> 156 <211> 23 <212> PRT
<213> Escherichia coli <400> 156 Leu Tyr Ser Arg Ala Ile Ser Ala Ala Phe Thr Ala Arg His Lys Gly Lys Asn Gin Ile Gin Phe Phe <210> 157 I, v , <211> 26 <212> PRT
<213> Escherichia coil <400> 157 Met Arg Phe Pro Val Ser Asp Leu Pro Asn Lys Ala Leu Leu met Glu Met Leu Glu Gin Val Val Ala Arg Lys Gin <210> 158 <211> 12 <212> PRT
<213> Escherichia coil <400> 158 Ala Leu Met Ile Ile Thr Cys Glu Thr Leu Arg Asp <210> 159 <211> 27 <212> PRT
<213> Escherichia coil <400> 159 Ala Gly Val Leu Lys Glu Ala Gin Arg Glu Ile Leu Leu Leu Thr Leu val Glu Lys Leu Lys Ser Val Leu Ser Pro Arg <210> 160 <211> 17 <212> PRT
<213> Escherichia coil <400> 160 Leu Ala Gin Ile Ser Gly Tyr Asp Phe Ala Val Ile Ala Asn Gly val Gin <210> 161 <211> 19 <212> PRT
<213> Escherichia coil <400> 161 Trp His Ala Ile Thr Leu Gly Gin Gin val Leu Thr Ile Met Ser Glu Arg Leu Pro <210> 162 <211> 17 <212> PRT

<213> Escherichia coli <400> 162 Gin Leu Arg Pro His Cys Ser Ile Gly Val Ala Met Phe Tyr Gly Asp Leu <210> 163 <211> 23 <212> PRT
<213> Escherichia coli <400> 163 Leu Tyr Ser Arg Ala Ile Ser Ala Ala Phe Thr Ala Arg His Lys Gly Lys Asn Gin Ile Gin Phe Phe <210> 164 <211> 26 <212> PRT
<213> Escherichia coli <400> 164 Met Arg Phe Pro Val Ser Asp Leu Pro Asn Lys Ala Leu Leu Met Glu met Leu Giu Gin Val val Ala Arg Lys Gin <210> 165 <211> 12 <212> PRT
<213> Escherichia coli <400> 165 Ala Leu Met Ile Ile Thr Cys Giu Thr Leu Arg Asp <210> 166 <211> 27 <212> PRT
<213> Escherichia coli <400> 166 Ala Gly val Leu Lys Glu Ala Gin Arg Glu Ile Leu Leu Leu Thr Leu Val Glu Lys Leu Lys Ser Val Leu Ser Pro Arg <210> 167 <211> 17 <212> PRT
<213> Escherichia coli <400> 167 Leu Ala Gin Ile Ser Gly Tyr Asp Phe Ala val Ile Ala Asn Gly Val Gin <210> 168 <211> 19 <212> PRT
<213> Escherichia coli <400> 168 Trp His Ala Ile Thr Leu Gly Gin Gin Val Leu Thr Ile Met Ser Glu Arg Leu Pro <210> 169 <211> 17 <212> PRT
<213> Escherichia coli <400> 169 Gin Leu Arg Pro His Cys Ser Ile Gly Val Ala Met Phe Tyr Gly Asp Leu <210> 170 <211> 23 <212> PRT
<213> Escherichia coli <400> 170 Leu Tyr Ser Arg Ala Ile Ser Ala Ala Phe Thr Ala Arg His Lys Gly Lys Asn Gin Ile Gin Phe Phe

Claims (66)

What is claimed:

1. A vector comprising at least one cyclic di-nucleotide synthetase enzyme gene.

2. The vector of claim 1, which is a gene-therapy vector.

3. The vector of any one of claims 1-2, wherein the vector is selected from the group consisting of adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus.

4. The vector of any one of claims 1-3, which is a DNA-based vector.

5. The vector of any one of claims 1-3, which is an adenoviral vector.

6. The vector of claim 5, which is a replication defective adenoviral vector.

7. The vector of any one of claims 1-6, wherein the at least one cyclic di-nucleotide synthetase enzyme gene is derived from a bacterial, fungal, protozoal, viral, or pathogenic strain.

8. The vector of claim 7, wherein the at least one cyclic di-nucleotide synthetase enzyme gene is derived from a bacterial strain.

9. The vector of claim 8, wherein the bacterial strain is Vibrio cholerae.

10. The vector of any one of claims 1-9, wherein the at least one cyclic di-nucleotide synthetase enzyme gene is selected from the group consisting of diadenylate cyclase (DAC), DncV, Hypr-GGDEF, DisA, cGAS, and diguanylate cyclase (DGC).

11. The vector of claim 10, wheirein the at least one cyclic di-nucleotide synthetase enzyme gene is DGC.

12. The vector of claim 6, wherein the DGC comprises a sequence which is at least 80%
identical to the sequences set forth in Table 1.

13. The vector of any of claims 11-12, wherein the DGC gene is VCA0956 gene.

14. The vector of claim 13, wherein the VCA0956 gene comprises a nucleotide sequence which is at least 80% identical to SEQ ID NO: 33.

15. The vector of any of claims 11-12, wherein the DGC gene is VCA0848 gene.

16. The vector of claim 15, wherein the VCA0848 gene comprises a nucleotide sequence which is at least 80% identical to SEQ ID NO: 68.

17. The vector of any of claims 1-16 which comprises an adenovirus selected from non-human, human adenovirus serotype, or any adenovirus serotype developed as a gene transfer vector.

18. The vector of claim 17, wherein the non-human adenovirus comprises an adenovirus selected from chimp, equine, bovine, mouse, chicken, pig, or dog.

19. The vector of claim 17, wherein the adenovirus is human adenovirus serotype 5.

20. The vector of claim 19, wherein the adenovirus has at least one mutation or deletion in at least one adenoviral gene.

21. The vector of claim 20, wherein the adenoviral gene is selected from the group consisting of E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5.

22. The vector of claim 21, wherein the adenovirus has a deletion in E1A, E1B, and E3, or combinations thereof.

23. The vector of any one of claims 1-22, wherein the at least one cyclic di-nucleotide synthetase enzyme gene is operatively linked to a transcriptional and translational regulatory sequences.

24. A combination comprising the vector of any one of claims 1-23.

25. The combination of claim 24 further comprising at least one therapeutic agent.

26. The combination of any one of claims 24-25, wherein the agent is another vaccine, an immunemodulatory drug, a checkpoint inhibitor, or a small molecule inhibitor.

27. The combination of any one of claims 24-26 further comprising a therapy for immune events.

28. The combination of claim 27, where the therapy is irradiation.

29. A pharmaceutical composition comprising the vector of any one of claims 1-23, and a pharmaceutically acceptable composition selected from the group consisting of excipients, diluents, and carriers.

30. The pharmaceutical composition of claim 29, wherein the pharmaceutical composition comprises the vector at a purity of at least 75%.

31. An adjuvant comprising the vector of any one of claims 1-23.

32. A vaccine comprising the vector of any one of claims 1-23, the pharmaceutical composition of any one of claims 29-30, or the adjuvant of claim 31.

33. The vaccine of claim 32 further comprising an antigen.

34. The vaccine of any one of claims 32-33, wherein the antigen is provide in a second adenoviral vector.

35. The vaccine of any one of claims 32-34, wherein the antigen is immunogenic.

36. The vaccine of any one of claims 32-35, wherein the antigen is an extracellular antigen.

37. The vaccine of any one of claims 32-36, wherein the antigen is a viral-associated antigen, pathogenic-associated antigen, protozoal-associated antigen, bacterial-associated antigen, fungal antigen, or tumor-associated antigen.

38. The vaccine of any of claims 32-37, wherein the antigen is selected from the group consisting of Ovalbumin (OVA)-specific, HIV-1-derived Gag Ag, Clostridium difficile-derived toxin B, and Clostridium difficile-derived toxin A.

39. A method of inducing or enhancing an immune response in a mammal, comprising:
administering to the mammal a pharmaceutically effective amount of the vaccine of any one of claims 32-38 such that the immune response is enhanced or stimulated.

40. A method of treating a mammal having a condition that would benefit from upregulation of an immune response comprising administering to the subject a therapeutically effective amount of the vaccine of any one of claims 32-38 such that the condition that would benefit from upregulation of an immune response is treated.

41. The method of any one of claims 39-40, further comprising administering one or more additional compositions or therapies that upregulates an immune response or treats the condition.

42. The method of claim 41, wherein the one or more additional compositions or therapies is selected from the group consisting of anti-viral therapy, immunotherapy, chemotherapy, radiation, and surgery.

43. The method of any one of claims 40-42, wherein the condition that would benefit from upregulation of an immune response is selected from the group consisting of cancer, a viral infection, a bacterial infection, fungal infection, and a protozoan infection.

44. The method of any one of claims 39-43, wherein the immune response is the innate immune response, adaptive immune response, or humoral immune response.

45. The method of any one of claims 39-44, wherein the vaccine increases or stimulates cyclic di-GMP (c-di-GMP), cyclic di-AMP (c-di-AMP), cyclic GMP-AMP (cGAMP), any cyclic di-nucleotide, or combinations therof,levels in said mammal.

46. The method of any one of claims 39-45, wherein the vaccine increases or stimulates the secretion of cytokines and chemokines.

47. The method of claim 46, wherein the cytokines and chemokines are selected from the group consisting of IFN-.beta., IL-1.alpha., IL-4, IL-6, IL12-p40, IFN-.gamma., G-CSF, Eotaxin, KC, MCP-1, MIP-1.alpha., MIP-1.beta., and RANTES.

48. The method of claim 44, wherein the vaccine increases or stimulates an immune response selected from the group consisting of DC maturation, NK cell response, T-cell response, and B-cell reponse, or combination thereof.

49. The method of claim 48, wherein the immune response increases the population of immunce cells selected from the group consisting of CD86+ CD11c+CD11b-DCs, CD69+
NK1.1+ CD3- NK cells, CD69+ CD19+ CD3- B cells, CD69+ CD3+ CD8- T cells, and CD69+
CD3+ CD8+ T cells, or combinations thereof.

50. The method of any one of claims 39-49, wherein the subject is a mammal.

51. The method of claim 50, wherein the mammal is an animal model of the condition.

52. The method of claim 50, wherein the mammal is a human.

53. The method of claim 50, wherein the mammal is an avian species.

54. The mthod of claim 54, wherein the avian species is G. gallus, or eggs derived therefrom.

55. The method of any one of claims 39-52, wherein the vaccine is administered intradermally, intramuscularly, intraperitoneally, intratumorally, peritumoroally, retroorbiatlly, or intravenously via injection.

56. The method of any one of claims 39-55, wherein the vaccine is administered concomitantly or conjointly.

57. The method of claim 56, wherein the first vector comprising the cyclic di-nucleotide synthetase enzyme gene lowers the effective dose for the second vector comprising the antigen.

58. The method of any one of claims 39-57, wherein the administration is repeated at least once.

59. The method of any one of claims 39-58, wherein the effective amount is from about 1x10 6 vp to about 5x10 11 vp.

60. The method of any one of claims 59, wherein the effective amount is from about 1x10 6 vp to about 5x10 9 vp.

61. The method of claim 60, wherein the effective amount is about 1x106 vp, about 1x10 7 vp, about 1x10 8 vp, or about 5x10 9 vp.

62. The method of claim 61, wherein the effective amount is about 5x10 9 vp.

63. The method of claim 59, wherein the effective amount is about 1×10 16, about 0.5x10 11, about 1x10 11, about 2x10 11, about 3x10 11, about 4x10 11, or about 5x10 11 viral particles (vp).

64. The method of claim 63, wherein the effective amount is about 2x10 11 vp.

65. The method of any one of claims 39-58, wherein the effective amount is about 10 µg/mL, about 20 µg/mL, about 30 µg/mL, about 40 µg/mL, about 50 µg/mL, about 60 µg/mL, about 70 µg/mL, about 80 µg/mL, about 90 ug/mL, about 100 µg/mL, about 125 µg/mL, about 150 µg/mL, about 175 µg/mL, and 200 µg/mL

66. The method of claim 65, wherein the effective amount is about 100 µg/mL.